Treatment of Streptozotocine Induced Diabetes Mellitus in Male Rats by Immunoisolated Transplantation of Purified Langerhans Islet Cells
B. Lame Rad,
Induction of experimental diabetes mellitus is indeed, the first step in the process of purification of pancreatic Langerhans islet cells of normal rats for transplantation under the testis subcutaneous of experimentally induced diabetic rats. For induction of experimental diabetes in male adult rats weighting 250-300 g (75-90 days), 60 mg kg-1 of streptozotocin was injected intravenously. Biopsy of pancreas tissue of diabetic and normal rats showed that the Langerhans islet beta cells of diabetic rats have been clearly degenerated. In the process of purification of islet cells, after collagenase digestion of pancreases, islets were isolated and dissociated; employing enzymes like DNase and trypsine, so the islet cells were changed into single cells and these cells were assayed by flow cytometry. Flow cytometry of these cells indicated that there were 91% of beta cells in cell suspension. Donor tissue in each step of this research was prepared from 38 adult wistar male rats. Transplantation was done in rats after 2-4 weeks induction of diabetes. Encapsulation of pancreatic islet cells allows for transplantation in the absence of immunosuppression. This technique which is called immunoisolation is based on the principle that transplanted tissue is protected for the host immune system by an artificial or natural membrane. The diabetic, treated and normal animals were kept in the metabolic cages separately and their body weight, consumption of food and water, urine volume, the levels of serum glucose, insulin and C-peptide quantities in all animals were measured. Analysis of variance showed a high significant difference between them.
to cite this article:
A. Akbarzadeh, D. Noruzian, SH. Jamshidi, A. Farhangi, M.R. Mehrabi, B. Lame Rad, M. Mofidian and A. Allahverdi, 2007. Treatment of Streptozotocine Induced Diabetes Mellitus in Male Rats by Immunoisolated Transplantation of Purified Langerhans Islet Cells
. Asian Journal of Biochemistry, 2: 31-41.
Diabetes is a chronic disease that is relatively common throughout the world.
In recent decades, various epidemiological studies have been carried out on
prevalence of diabetes mellitus in Iran, according to which the population of
diabetics was estimated to exceed 1.5 million. In 2004, according to the World
Health Organization reports, more than 150 million people throughout the world
suffered from diabetes (World Health Organization; http://www.who.int/medacenter/factsheets/fs/138/en/Page1-3).
The only simple, inexpensive, easy and available way is to refine the Langerhans
islets and to graft them under the testis subcutaneous, which we hope to present
the progressive stages of this method. Experimental diabetes mellitus has been
induced in laboratory animals by several methods. The generally effective method
is to take the pancreas out of the body. However, to induce a notable form of
diabetes, at least 90-95% of the pancreas has to be removed. Otherwise, the
Langerhans islets cells in the remaining pancreas may undergo hypertrophy and
secrete sufficient amount of insulin for fulfilling the natural metabolic needs.
The second method for creating diabetes in animals is injection of drugs such
as alloxan or streptozotocin. These materials inflate and ultimately, degenerate
the Langerhans islets beta cells (Ikebukuro et al., 2002; Weiss, 1982).
A less reliable method for creating diabetes is the injection of the anterior
hypophysis extract (Gray and Morris, 1987; Sutherland et al., 2001).
The final symptoms of insulin deficiency are clearly seen in rats afflicted
with diabetes chemically by streptozotocin (Pipeleers et al., 1991a,
1985). Injection of 60 mg kg-1 of body weight of streptozotocin results
in the toxicity of beta cells and emergence of clinical diabetes, within 2-4
days. Therefore, the study made us, first, to induce experimental diabetes mellitus
in order to study the effect of transplantation of the Langerhans islets beta
cells in diabetic rats with streptozotocin so as to be able to study the clinical
parameters before and after the pancreas islet cells transplantation (Lacy and
Kostianovsky, 1967; Olack et al., 1999). Transplantation of pancreas
components can be done in one of the following forms: i) Transplantation of
dissociated Langerhans islets beta cells; ii) Transplantation of mass of the
Langerhans islets cells; iii) Transplantation of embryonic tissues; iv) Transplantation
of neonatal tissues; v) Transplantation of pancreas for treatment of diabetes
mellitus, which has been carried out successfully in different areas such as
liver, kidneys, spleen, testis. However, transplantation of the Langerhans islets
beta cells as a logical solution for the treatment of these patients is still
argued (Winkel, 1982; Rabinovitch et al., 1982). Transplantation of the
Langerhans islets cells is a new method for the treatment of diabetes. The standardized
and optimized separation and purification conditions of Langerhans islets cells
is one of the most important phases of the transplantation (Boker et al.,
2001; Yasumizu et al., 1987). It is inexpensive, simple, safe and practical
treatment method for all diabetic patients. Factors such as the number of implanted
cells, capacity of performance of the new medium and the size of cell groups
are effective in the relative control of the metabolism after transplantation
which is needed to be considered in such studies (Rastellini and Shapiro, 1997;
Holemans et al., 1997).
Materials and Methods
Collagenase, Crystalline Trypsin, Bovine Pancreatic DNase, 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-Ethan-sulfonic
acid (HEPES), silicon dicholorodimethylsilan and bovine serum albumin fraction
V were supplied from the German Company, Merck. Percol is a commercial solution
from silicon particles, coated with polyvinyl pyrolidone. Silicon coated with
polyvinyl pyrolidone is used for sterilized cellular separation. Streptozotocin
was supplied from the Swedish company Pharmacia. Ethylene glycol-bis (β-amino
ethyl ether)-N, N, N, N,-tetra acetic acid are products of Sigma.
All mediums were sterilized by 0.22 micrometer filters and the materials
were autoclaved or were purchased as sterilized single use materials. The glass
containers used for collecting Langerhans islets cells were siliconized with
silicon. Silicon-coating consists of 30 min incubation of the containers to
be used with sterilized solution of 10 μg mL-1 silicon after
washing with distilled water. Separation of the islet and cellular purification
were carried out in an Embryo Handling buffer (EH buffer) medium consists of
the following components: 123 mM NaCl, 1.8 mM CaCl2, 0.8 mM MgSO4,
5.4 mM KCl, 1.0 mM NaH2PO4, 4.2 mM NaHCO3,
2.8 mM Glucose, 10 mM HEPES. The medium was completed with 2.5 and 5% solutions
from fraction V Bovine Serum Albumin (BSA), its pH was kept at 7.3 with 5% CO2
in room temperature and the final volume was reached to 1 L.
Six adult Wistar rats weighting 250-300 g (75-90 days old) were used for
inducing diabetes and 6 rats were taken as control. The donor tissues were taken
from 38 male adult Wistar rats weighting 250-300 g (75-90 days old). The Islet
beta cells were purified from pancreas of 38 rats and transplanted to 19 rats
and the other 19 rats were taken as control.
Induction of Diabetes in Rats
The animals were injected with streptozotocin at the dose of 60 mg kg-1
of the body weight intravenously. Streptozotocin induces diabetes within 3 days
by destroying the beta cells (Ikebukuro et al., 2002). Diabetic animals
and non-diabetic control group were kept in metabolic cages individually and
separately and under feeding and metabolism control (Elias et al., 1994).
Glucose in the blood of diabetic rats exceeded that of the non-diabetic control
ones (Weiss, 1982). Food consumption was measured in terms of (g), water consumption
was measured in terms of (mL) and urine volume was measured in terms of (mL)
on a daily basis while every 2-4 weeks in 80 days the levels of C-peptide, insulin
and glucose in blood serum were also measured, so that chemical diabetes was
verified in rats injected with streptozotocine (Pipeleers et al., 1985;
Holemans et al., 1997).
Pancreatic and Testis Biopsy of Normal and Diabetic Rats
For the study and comparison of pancreas Langerhans islet beta cells testis
tissue in diabetic rats induced by streptozotocine and normal rats, pancreatic
and testis biopsy of normal and diabetic rats were done and samples were fixed
in 10% formalin and were given to the department of electronic microscope for
light microscopic examination. After framing in paraffin, thin 3-micron tissue
cuts were created. Staining was carried out by Hematoxylene and Eosin stain
in order to recognize the normal and diabetic rats pancreas tissue. Figure
1 and 2 show pancreatic biopsy of normal and diabetic
rats with Leitz microscope by 4000 times enlargement. The comparison of these
pictures shows that the tissue of Pancreas and Langerhans and the beta cells
of diabetic rats have been degenerated irreversibly (Fig. 1
and 2), while no changes are observed in the testis tissue,
under the subcutaneous of the normal and diabetic rats (picture has not been
Separation of the Langerhans Islets
The pancreatic Langerhans islets cells were separated from the rats with
the modified collagenase digestion method (Ikebukuro et al., 2002; Sutherland
et al., 2001). Two hours prior to dissection for identifying the pancreas,
pilocarpine (0.2 mL from 0.2% solution) was injected to the animals intraperitoneally
(Pipeleers, 1981; Gray and Morris, 1987). To carry out dissection, first the
animals were anesthetized in an appropriate desiccator. Then, by opening the
rats abdomen and closing the pancreatic canal, 10 mL of Embryo Handling
(EH) buffer containing 1.5 mg mL-1 of collagenase was injected into
the pancreas to expand it and to make the lymphatic nodes and fatty tissues
of the pancreas separable. Then, it was cut into pieces (Pipeleers et al.,
1991a). After 15 sec sedimentation, the upper solution was disposed off and
the tissue suspension was diluted with an equal volume of Embryo Handling (EH)
buffer, including 4 mg mL-1 of collagenase (Lacy and Kostianovsky,
1967). The tissue was shaken at 37°C for 10 min at 300 rpm so as to be digested.
Then, the Langerhans islet beta cells were separated in room temperature for
3 min by mildly pipetting (Olack et al., 1999). What is obtained from
digestion is then passed through a nylon sheet of 500 μm diameter. The
filtered part was centrifuged for 3 times and each time it was washed with Embryo
Handling (EH) buffer and further made into a suspension in Embryo Handling (EH)
solution (Boker et al., 2001). What remained on the filter was further
made into suspension in Embryo Handling (EH) solution without collagenase and
then shaken in the shaker incubator for 4 min at 300 rpm at 37°C and filtered
as mentioned above. The product of the second digestion was finally washed in
EH solution. The Langerhans islets were filtered and washed and the remained
parts were carefully examined by light microscope and the cleaned islet beta
cells were collected (Fig. 3). This method should be carried
out appropriately, carefully and rapidly so that the islets would be less damaged
(Gray and Morris, 1987). The Langerhans islets suspension was first kept in
room temperature for 8 min and then aspired by a 9 inch siliconized Pasteur
pipette. Then, trypsin with final concentration of 25 μg mL-1
and DNase with final concentration of 1.5 μg mL-1 were added
to it (Pipeleers et al., 1991b). The degree of enzymatic differentiation
and dissociation were regularly examined with contrast-phase microscope and
when 50-60% of the cells converted into single units, the work was stopped (Yasumizu
et al., 1987; Rastellini and Shapiro, 1997). This condition often occurs
after 10 min. The suspension of the Langerhans islets cells was diluted immediately
with 40 mL of Embryo Handling buffer and the whole collection was put in ice
and filtered by passing through a 63 μm diameter nylon sheet. Thus, the
undigested materials and the big cell masses were eliminated. The resulting
product, which contained single cells, was centrifuged for 6 min in 300 g. (Titus
et al., 2000; Thomas et al., 1999). The sediment was further changed
to suspension and centrifuged. In this stage, the cellular sediment was suspended
in isotonic percol solution with density of 1.040 g mL-1 and was
put in ice for 10 min so that the cellular suspension was layered and thus,
the dead and destroyed cells and cell pieces obtained in consecutive centrifugation
were eliminated. Finally, in the cellular suspension layer, the healthy cells
were dissolved in the physiological serum (Holemans et al., 1997).
||Pancreatic biopsy of normal rats
||Pancreatic biopsy of diabetic rats that confirms the destruction
of islets and cells due to the effect of streptozotocin
||The existing cells of Langerhans islets at the cell suspension
colored by the Gimsa and photographed by Litz microscope, by 1000 time magnification
This system is a new technique, through which the physicochemical specifications
of the cells or any biological component are recorded individually when they
pass against laser beam. The individuality and solution nature of the cells
are important in flow cytometry. The sample must be a solution from the outset
or be made into a solution with enzymatic methods, in which each tissue is prepared
with special methods of its own. Measurement of parameters such as size, form,
DNA content, surface cell receptors, enzymatic activity, membrane permeability
and calcium pump are possible with this method (Nielsen et al., 1982).
Our goal in flow cytometry is to obtain information on the homogeneity of beta
cells and the percentage of homogeneity of these cells in cellular suspension
obtained at the end of purification of the Langerhans islets cells so as to
determine the percentage of beta cells in the transplanted suspension. In view
of the considerable difference in the sizes of types of Langerhans islets cells,
a sample of cellular suspension solution can be injected into the flow cytometry
system and prepare the appropriate graph, which indicates the types of cells
and their percentage in the suspension. The Langerhans islets cells purified
by the collagenase method were 91% of beta cells in the cellular suspension
(Winkel et al., 1982; Rabinovitch et al., 1982) (Fig.
||Superficial distribution curve of Langerhans islets cells
suspension obtained from flow cytometry, in the flow cytogram of a homogenous
bulk of cells with purity of 91% which belongs to the cells with fewer granularities
among the langerhans islets cells, i.e., beta cells
Islets were specifically stained by dithizone. 10 mg dithizone was dissolved
in absolute ethyl alcohol (50 mL concentrated NH4OH), supplemented
with 12 mL Hanks solution Sigma [45 mM Na2HPO4,
2.5 mM citric acid, 0.1% Triton X-100]. Just before using, the preceding solution
was diluted with Hanks solution (pH 7.8) by 1 to 100, passed through a
0.22 mm filter membrane. Islet suspension was mixed with dithizone and placed
10 min and identified by light microscope (Pipeleers and Pipeleers-Marichal,
β-cells were fixed using Bruins solution [71.4% picric acid solution
(1.2% w/v), 23.8% formalin and 4.8% glacial acetic acid]. After 24
h, cells were rinsed three times with PBS, dehydrated and permeabilized
with graded concentrations of ethanol and incubated for 2 h at room
temperature with an anti-insulin antibody (Sigma-Aldrich. Com) diluted
1:1,000 in PBS. After rinsing, slides were incubated for 1 h at room
temperature with a fluorescein-labeled goat anti-guinea pig second
antibody (1:400). After rinsing in PBS, slides were covered with
0.02% p-phenylenediamine in PBS-glycerol (1:2, v/v) and screened
by fluorescence microscopy (Pipeleers et al., 1991b).
Treatment of Diabetic Rats by Transplantation of Langerhans Islets Cells
The purified cells of the Langerhans islets were transplanted to diabetic
rats stimulated with streptozotocin in a group of diabetic samples inside the
testis subcutaneously (Ikebukuro et al., 2002; Boker et al., 2001;
Gray and Morris, 1987; Pipeleers et al., 1991a; Sutherland et al.,
2001). Transplantation of the cellular suspension in the physiological serum
was carried out by using needle No. 20, inside the testis, subcutaneously in
each injected rat. Figure 4 successfully shows the transplanted
Langerhans islets in diabetic rat (Yasumizu et al., 1987; Lanza and Ecker,
Measurement of Glucose, Insulin and C-Peptide in Blood
Normal, diabetic and donor rats were anesthetized with ether (two minutes
contact with ether does not affect blood glucose, insulin or C-peptide concentrations).
Each time 0.5 mL of blood was taken from them in order to measure sugar, insulin
and C-peptide (Pipeleers et al., 1985). Blood was taken from the heart.
The samples were collected in sterilized tubes and kept at 4°C and after
separating the clot, the serum was separated by centrifuging (Van De Winkel
and Pipeleers, 1983). Blood glucose was measured by glucose oxidase method and
serum C-peptide by radioimmunoassay method. This phase of the study was carried
out once every 2-4 weeks for 80 days in diabetic and donor rats as well as in
their control counterparts (Lanza and Ecker, 1999).
Biopsy and Histology of Langerhans Islets Cells Growing
Two months after transplantation, the transplanted areas were vivisected
in order to identify the Langerhans islets cells grown in the transplant receptor.
For this purpose, the testis of the recipient rats were removed, stabilized
in 10% formalin and given to the Electronic Microscope Department for light
microscopic examination. After framing in paraffin, thin 3-micron tissue cuts
were created. Staining was carried out by Hematoxylene and Eosin stain in order
to recognize the transplanted islets cells. Figure 4 shows
transplanted Langerhans islets cells normal growing with Leitz microscope by
4000 times enlargement under the testis subcutaneous of treated rat.
Normal levels of glucose, insulin and C-peptide in healthy adult rats were
measured to be 135±5 mg dL-1, 2.1±0.2 m IU L-1
and 0.056±0.001 ng mL-1, respectively. Daily consumption of
water and food in healthy adult rats were found to be 30±5 mL and 10±2
g, respectively. Daily urine volume in healthy adult rats was 10±1 mL.
In diabetic rats the levels of glucose, insulin and C-peptide were measured
as 500±20 mg dL-1, 1.5±0.2 m lU L-1 and
0.042±0.002 ng mL-1, respectively. Daily consumption of water
and food in them were measured as 145±5 mL and 45±4 g.
||Shows data of number, weight, age, amount of streptozotocine injection,
glucose, insulin, C-peptide of blood, consumption of water, food and volume
of urine in normal, diabetic and treated Rats
||The continuous changes in average of body weight in 8 non-adult
rats in three healthy, diabetic and treated phases
||A comparison of the curves relating to the average changes
in 19 body weight in the three groups of healthy, diabetic and transplanted
rats. These curves reveal loss of weight and thinness due to streptozotocin
used for diabetes induction in adult rats and elimination of these effects
after transplantation of pancreatic Langerhans islets cells during 80 days
Daily urine volume in diabetic rats was measured as 130±5 mL. Levels
of glucose, insulin and C-peptide in treated adult rats were measured as 145±10
mg mL-1, 2±0.2 m IU L-1 and 0.053±0.009
ng mL-1, respectively. Daily consumption of water and food in treated
adult rats were measured as 40±5 mL and 30±5 g, respectively.
Daily urine volume in treated adult rats was measured as 35±5 mL (Table
1). Changes of weight in adult and non-adult diabetic rats vary. Since the
non-adult rats are in the growing age, diabetic loss of weight is not seen in
them and they even show a slight weight gain. Figure 5 shows
continuous changes in average of body weight in 8 non-adult rats in three healthy,
diabetic and treated phases and a slight increase in the weight of non-adult
rats in the three healthy, diabetic and treated phases. But in adult rats, however,
diabetes is accompanied by loss of weight. Two to four weeks after diabetes
induction and observing its effects, transplantation of purified Langerhans
islets cells was carried out by healthy rats pancreas with modified collagenase
digestion method with 91% beta cells in the cellular suspension for treatment
of diabetes. Fig. 6 shows the average changes in the body
weight of 19 diabetic rats treated by transplantation of Langerhans islets cells
and normal ones during 80 days.
||The changes of average level of glucose in serum of 19 diabetic
rats treated by transplantation of Langerhans islets cells and normal ones
during 80 days
||The average in changes in the level of insulin in serum of
19 diabetic rats treated by transplantation of Langerhans islets cells and
normal ones during 80 days
weight of 19 diabetic rats treated by transplantation of Langerhans islets
cells and normal ones during 80 days. This diagram reveals the loss of weight
and thinness due to streptozotocin used for diabetes induction in adult rats
and elimination of these effects after transplantation of pancreatic Langerhans
islets cells. By analyzing of variance (ANOVA) with SPSS. 12, the Standard Error
Mean (SEM) is found to be 8.19, F = 40.87, df = 2, 57, p<0.001, which well
indicates the weight loss in diabetic adult rats. By carrying out this operation,
signs of recovery were gradually observed in the rats, so that the levels of
glucose, insulin and C-peptide in transplanted rats were 145±11.2 mg
dL-1, 2±0.2 m IU L-1 and 0.053±0.009 ng
mL-1, respectively. Fig. 7 shows the average of
the changes in the level of glucose in blood serum of 19 diabetic rats treated
by transplantation of Langerhans islets cells and normal ones during 80 days,
that by using ANOVA on transplanted and diabetic rats, we found the standard
error mean (SEM) to be 48.1, F = 903.18, df = 2, 11, p<0.001, which well
indicates the glucose loss in diabetic adult rats. By carrying out this operation,
signs of recovery were gradually observed in the rats. Figure
8 shows the average changes of insulin in blood serum of 19 diabetic rats
treated by transplantation of Langerhans islets cells and normal ones during
80 days, in which the ANOVA on transplanted and diabetic rats shows that the
standard error mean (SEM) is equal to 0.088, F = 8.53, df = 2, 12, p<0.005,
which well indicates the insulin loss in diabetic adult rats. By carrying out
this operation, signs of recovery were gradually observed in the rats. Figure
9 shows the average of the changes in the level of C-peptide in blood serum
of 19 diabetic rats treated by transplantation of Langerhans islets cells and
normal ones during 80 days, in which the ANOVA on transplanted and diabetic
rats shows that the standard error mean (SEM) is equal to 0.002, F = 4.85, df
= 2, 12, p<0.029, which well indicates the C-peptide loss in diabetic adult
rats. By carrying out this operation, signs of recovery were gradually observed
in the rats consequently, data analysis of glucose, insulin and C-peptide show
the high significant difference between transplanted and diabetic rats serum
and confirm the success of the transplantation project. Moreover, the daily
consumption of water and food reached to the relatively normal limit of 40±5
cc and 30±5 g, respectively and daily urine in treated rats was measured
as 35±5 cc. Pancreatic biopsy of normal and diabetic rats confirmed that
the islet cells were destroyed due to the effect of streptozotocin in diabetic
rats. The comparison of pancreatic biopsy of normal and diabetic rats shows
that the tissue of Pancreatic Langerhans and the beta cells of diabetic rats
have been degenerated irreversibly, while no change is observed in the tissue
under the testis subcutaneous of the normal and diabetic rats.
||The changes of average level of C-peptid in serum of 19 diabetic
rats treated by transplantation of Langerhans islets cells and normal ones
during 80 days
||The existing transplanted Langerhans islet cells under the
testis subcutaneous of the diabetic rat which has been treated via transplantation
of Langerhans islet cells. Observed after taking a tissue from the inside
the testis subcutaneous and stabilizing in 10% formaldehyde solution
The diabetic rats did not have a natural living or natural length of life while
the healthy and treated rats had a natural life. The diabetic rats gradually
lost their eyesight while the healthy and treated rats had natural vision till
the end of their life. No discrepancy was seen between the results of our study
and those of other researchers. The only newly found, inexpensive and available
method now is transplantation of Langerhans islets cells for the treatment of
insulin-dependent diabetes mellitus since it is inexpensive, easy to do, available
and effective. One of the most important phases of transplantation is standardization
of the conditions of dissociation and purification of the Langerhans islets
cells. This is easy to do and practical and the researchers, after achieving
and stabilizing such a method, will be able to save the diabetic patients by
transplanting the Langerhans islets and solve the problem of more than 1.5 M
diabetic patients in Iran and 200 M diabetic patients throughout the world.
However, the method of transplantation of the Langerhans islets inside the testis
subcutaneous of diabetic rats (Fig. 10) successfully shows
the transplanted Langerhans islets, i.e., in an immunity-quarantined space,
it prevents access of the immunity cells to the external transplanted cells
and prevents rejection of the transplantation. The rat is cured 100% as a result
of secretion of the transplanted Langerhans islets cells under the testis subcutaneous.
In this method, approximately 5,000 islets are needed for each kg of rat body
weight. This method is important because of its simplicity and accessibility.
Streptozotocin prevents DNA synthesis in mammalian and bacterial cells. Streptozotocin, which is used intravenously by rapid injection or constant short diffusion, stimulates the tissues. Metabolically, a slight deviation of the glucose-bearing pain from the normal limit has been seen in patients treated with a certain dose of streptozotocin, which is generally reversible. However, the insulin shock, which is one of its other effects, is irreversible (Weiss, 1982; Ikebukuro et al., 2002). In this study, the clinical manifestations and also the amount of glucose, insulin and C-peptide after using a 60 mg kg-1 dose of streptozotocin, ensured induction of diabetes in rats. Hyperglycemia, hypoinsulinemia, polyphagia, polyuria and polydipsia accompanied by weight loss were seen in adult rats within three days of streptozotocin treatment and within one week to ten days, the amounts of the relevant factors were almost stable, which indicates irreversible destruction of Langerhans islets cells moreover, Researchers around the world have used streptozotocin to create experimental diabetes because it is a simple, inexpensive and available method (Gray and Morris, 1987; Pipeleers et al., 1991a). Our results are similar with those of Ikebukuro (2002a) and Elias (1994). With the transplantation of the cellular suspension obtained, it was expected that, due to secretion of insulin by transplanted beta cells, the level of blood serum glucose would fall to the normal, healthy limit and the amount of insulin and C-peptide of the plasma would increase and the clinical manifestations of the disease, such as polyuria, polyphagia and polydipsia would be eliminated. All these were clearly seen immediately and completely in the day after transplantation. The considerable point in this research is that inbred rats were not used as receptor or donor of the transplantation. In spite of this, however, no sign of rejection of the transplantation was observed (Winkel et al., 1982). To explain this, one must say that the phenomenon of immuno-isolation due to the effect of transplantation of Langerhans islets cells, in an immunity-quarantined space, prevented access of the immunity cells to the external transplanted cells and rejection of the transplantation (Rabinovitch et al., 1982). Transplantation of Langerhans islets cells is generally used for treating a type of diabetes that results from the autoimmune destruction of beta cells of the islets. Therefore, as expected, this autoimmune process also continues with respect to the transplanted beta cells. In this research, by carrying out transplantation of Langerhans islets cells in parts of the body with a special immuno-isolated position, the risk of destruction of the transplanted beta cells by the autoimmune process in the transplantation receptor was completely eliminated (Yasumizu et al., 1987). The technique of transplantation of the Langerhans islets cells inside the testis, subcutaneously, in the absence of immunological inhibitors to support the transplanted tissue against the host immunity system is a new way of success in this path (Thomas et al., 1999; Rastellini and Shapiro, 1997). In this process, the islets can be encircled in a semi-permeable membrane that allows food and oxygen to reach to the Langerhans islets and the insulin to be released into the blood flow while, at the same time, it creates a mechanical barrier separating the potentially destructive immunity cells and the antibodies from the islets cells and thus preventing the rejection of the transplantation (Titus et al., 2000). Statistical data relating to F, df, values of p, food, water, urine, body weight and SEM in the entire test group, compared to the findings (Lanza and Ecker, 1999; Pipeleers et al., 1991; Gray and Morris, 1987) show the greater success of the transplantation of the Langerhans islets in rats as achieved in their work.
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