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Up-Regulation of the Expression of Activins in the Pancreatic Duct by Reduction of the ß-Cell Mass

Departments of Cell Biology and Molecular Medicine (Y.-Q.Z., H.Z., A.M., H.K., T.T., I.K.), Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan; and Department of Internal Medicine and Molecular Science (J.-i.M.), Graduate School of Medicine, Osaka University, Suita 565-0871, Japan

Address all correspondence and requests for reprints to: Itaru Kojima, M.D., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan. E-mail: ikojima{at}showa.gunma-u.ac.jp.

	Abstract

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Activins expressed in progenitor cells of the pancreas regulate differentiation of endocrine cells during development. Neogenesis of ß-cells takes place in adult animals under some conditions, and ß-cells are thought to arise from precursors locating in the pancreatic duct. In the present study, we investigated whether or not activins are expressed in the duct where ß-cell neogenesis is initiated. mRNA for the ß_A- and ß_B-subunits was expressed in isolated mouse pancreatic ducts. Immunohistochemically, the ß_A-subunit was detected in the pancreatic duct and colocalized with cytokeratin, a marker of ductal cells. The ß_A-subunit was also expressed in nestin-positive cells in the duct. Likewise, the ß_B-subunit was detected in the pancreatic duct. In addition, mRNA for the type II and type IIB activin receptors was expressed in the duct. Expression of mRNA for two activin subunits was markedly increased after streptozotocin injection. Similarly, the mRNA expression was up-regulated after partial pancreatectomy. These results indicate that activins are expressed in the pancreatic duct and are up-regulated shortly after the reduction of the ß-cell mass. Induction of activins in the duct may be a critical step in the initiation of ß-cell neogenesis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
ACTIVINS, MEMBERS OF the TGF-ß superfamily, regulate growth and differentiation of many types of cells in multiple biological systems (1, 2, 3), govern embryonic axial patterning, and modulate the function of foregut-derived organs (4, 5). We previously reported that activin A is expressed in epithelial cells in the pancreatic bud during development and, subsequently, in endocrine precursor cells expressing both insulin and glucagon (6). It is now known that activins promote the formation of the pancreatic bud, determine the differentiation of endocrine and exocrine cells in the pancreatic anlage, and induce the formation and migration of pancreatic islets (7, 8, 9, 10, 11, 12). Thus, activins regulate differentiation of pancreatic endocrine cells as an autocrine/paracrine factor during development. Formation of ß-cells is active after birth but markedly decreases shortly after weaning. ß-Cells turn over slowly in adult animals. However, when the ß-cell mass is reduced by several means, neogenesis of ß-cells takes place to compensate for the reduction of the ß-cell mass (13, 14, 15). Several lines of evidence indicate that progenitor cells still exist in adult pancreas. They are located in the pancreatic duct or in the vicinity of the duct and convert to endocrine cells in response to the differentiation signals (16, 17, 18). However, only limited information is currently available as to the mechanism regulating the initiation of ß-cell neogenesis. Specifically, it is not certain which of the differentiation factor(s) is involved in this important step. It is therefore interesting to determine whether or not activins are expressed in the progenitor cells of the adult pancreas and regulate their differentiation as they do during the development of the pancreas. In an attempt to address this issue, we measured the expression of activin subunits by using immunohistochemical techniques and RT-PCR. The results indicate that both ß_A- and ß_B-subunits of activins were expressed in ductal cells, and reduction of the ß-cell mass led to induction of the expression of activins in ductal cells. Given that activins regulate endocrine determination, induction of activins in ductal cells may be critical in the initiation of ß-cell neogenesis.

	Materials and Methods

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Materials
Polyclonal antibody to human activin A was raised in a rabbit, which recognizes activin A (ß_A-ß_A) and ß_A monomer but neither inhibin A (-ß_A) nor the ß_B-subunit (19). Polyclonal antinestin antibody was raised in a rabbit using a C-terminal oligopeptide (1602–1618) derived from human nestin as an antigen. Monoclonal mouse antihuman ß_B antibody was obtained from Oxford Bio-Innovation Ltd. (Oxford, UK). Polyclonal guinea pig antiporcine insulin antibody was provided by Dr. T. Matozaki (Gunma University, Maebashi, Japan). Polyclonal rabbit anticytokeratin (anti-CK) antibody was purchased from Nichirei Corp. (Tokyo, Japan). Rabbit polyclonal antifactor VIII/von Willbrand factor was purchased from Funakoshi (Tokyo, Japan). Polyclonal rabbit antiactivin type II receptor and antiactivin type IIB receptor antibodies were generously provided by Dr. K. Miyazono (University of Tokyo, Tokyo, Japan). Polyclonal goat antiactivin type II and type IIB receptor antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cy3-conjugated donkey antiguinea pig IgG and fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG and FITC-conjugated donkey antimouse IgG (1:500) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Other commercially available antibodies were from sources described previously (20).

Intraperitoneal injection of streptozotocin (STZ) and partial pancreatectomy
Male DDY mice (6–8 wk) were obtained from Imai Animal Company (Saitama, Japan). STZ (200 µg/g body weight) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) freshly dissolved in citrated buffer (pH 4.5) was injected ip into mice. Control animals were injected with equivalent volumes of citrate buffer. STZ-treated and control mice were later killed by decapitation at 6, 12, 24, 48, and 72 h. Mice were anesthetized by an ip injection of sodium pentobarbital (50 µg/g body weight). Partial pancreatectomy was performed by removing 50–60% of pancreas tissue according to Oberg-Welsh et al. (21). Sham operations were performed in mice of comparable age and weight. Pancreatectomized and sham-operated animals were subsequently kept together with free access to pelleted food and tap water before they were killed by decapitation at postoperational 6, 12, 24, 48, and 72 h. At each time point, the pancreas of STZ-treated mice or the remnant pancreas in partial pancreatectomized mice was removed and fixed with Bouin’s fixative before paraffin embedment. Fixed tissues were dehydrated in a graded ethanol series, cleared in xylene, and embedded in paraffin blocks. Sections were cut on a sliding microtome and then subjected to immunohistochemical procedures. Some of these pancreases were then incubated in the presence of collagenase (Funakoshi, Japan) for 10 min at 37 C, and the ducts were subsequently identified using stereo-microscopy and then hand-picked. The ducts were washed with PBS and then subjected to RNA isolation as described below.

To elevate the plasma glucose concentration, glucose (2 g/kg) was injected ip every 30 min for 2 h and changes in the plasma glucose concentration was measured. The plasma glucose concentration peaked at 3 h and the peak value was 426 ± 31.7 mg/dl (mean ± SE, n = 10). The plasma glucose then declined gradually and returned to the basal value by 6 h.

Relative quantification of activin mRNA using real-time RT-PCR
Pancreatic ducts were obtained after collagenase digestion (22). Ductules were picked up by inspection under microscopy. Intercalated ducts and centroacinar cells were not included. The expression of cytokeratin was confirmed by immunohistochemistry. Contamination of acinar cells and endocrine cells were checked by staining with antiamylase antibody or a mixture of antiinsulin and antiglucagon antibodies. More than 95% of the ducts were stained with anticytokeratin antibody and contamination of acinar cells and endocrine cells was negligible. Total RNA was extracted from the pancreatic ducts using TRIzol Reagent (Life Technologies, Inc., Grand Island, NY). mRNA was extracted using the Quick Prep Kit (Pharmacia LKB Biotechnology, Piscataway, NJ). RNA and mRNA samples were pretreated with deoxyribonuclease to remove contamination of genomic DNA. First-strand cDNA was synthesized by using the Preamplification System for First Strand cDNA Synthesis Kit (Life Technologies, Inc.). The details of the procedure were described previously (23). Oligonucleotide primers were 5'-AGCCAAACGGGTCATCATCTC-3' (sense) and 5'-TGCCTGCTTCACCACCTTCTTG-3' (antisense) for mouse glyceraldehyde phosphate dehydrogenase (GAPDH), 5'-TGCTGCACTTGAAGAAGAGACCC-3' (sense) and 5'-TGGTCCTGGTTCTGTTAGCCTTG-3' (antisense) for the mouse activin ß_A-subunit, and 5'-ATCGACTTTCGGCTCATCGG-3' (sense) and 5'-CACGATCATGTTGGGCACATC-3' (antisense) for the activin ß_B-subunit; 5'-GGGAAAGAGACAGAACCAACCAGAC-3' (sense) and 5'-TGGGCTGTG TGACTTCCATCTC-3' (antisense) for mouse activin type II receptor; 5'-GACTGGTGTTGAACCTTGCTATGG-3' (sense) and 5'-TGGGCTGTGTGACTTCCATCTC-3' (antisense) for mouse type IIB receptor; 5'-CAGGCTTTTGTCAAGCAGCAC-3' (sense) and 5'-TGGTGCAGCACTGAT CTACAATG-3' (antisense) for mouse insulin; 5'-GTTCATAACCCATC AAGACCTTGG-3' (sense) and 5'-TTGGATTG AGGTAACTTCCACAGG-3' (antisense) for mouse amylase, respectively. Real-time RT-PCR was performed using SYBR GREEN PCR Core Reagents (Applied Biosystems, Foster City, CA). Thermal cycling conditions were 2 min at 50 C and 10 min at 95 C followed with 40 cycles of 95 C for 45 sec, 60 C for 45 sec, 72 C for 45 sec. Data were collected using the ABI PRUSM 7700 SDS analytical thermal cycler (PE Biosystems, Foster City, CA). Reactions without cDNA were used as a negative control. Each sample was tested in triplicate. Aliquots of the PCR products were analyzed by agarose gel electrophoresis to check their quality and to ensure the correct size of the fragment. The standard curves for the ß_A- and ß_B-subunits and GAPDH were determined using serial dilution of the PCR product, respectively, ranging from 10⁵ to 10² copies. The ratio of DNA copies of the ß_A- or ß_B-subunit to GAPDH was calculated for their relative expressions.

Immunohistochemistry
The paraffin sections (2.5 µm) were deparaffinized, dehydrated in a graded ethanol series, and preincubated in normal horse or goat serum. To study the expression of activin and CK in the pancreas, consecutive sections were used. For double-staining, the sections were incubated with antibodies at room temperature for 1 h. After washing with PBS, they were then incubated with a mixture of Cy3-conjugated donkey antiguinea pig IgG and FITC-conjugated goat antirabbit IgG (1:500) and 4-diamidino-2-phenylindole (Roche Molecular Biochemicals, Mannheim, Germany) for 1 h at room temperature and then washed with PBS and coverslipped with PermaFluor Aqueous Mountant (IMMUNON SHANDON, Pittsburgh, PA). Immunofluorescence images were recorded with an Olympus Corp. for AX70 Epifluorescence microscope (Olympus Corp., Tokyo, Japan) equipped with a PXL 1400 cooled-charge-coupled device camera system (Photometrics, Tucson, AZ), which was operated with IP Lab Spectrum software (Signal Analysis, Vienna, VA). To detect the expression of the ß_B-subunit of activin in the pancreas, frozen slices were used. The pancreas was removed according to the experimental protocol, embedded in a Tissue-Tek OCT compound (Miles, Inc., Elkhart, IN) and frozen in liquid nitrogen. Frozen sections (8 µm) were cut with a Jung CM 3000 cryostat (Leica Corp., Wien, Austria), mounted on poly-lysine coated slides and fixed in 4% paraformaldehyde for 30 min at room temperature. For control, sections were incubated without the first antibody.

For detection of the activin ß_A-subunit, the avidin-biotin complex (ABC) method was also performed using Vextastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). The mounted sections were incubated for 1 h with rabbit polyclonal anti-ß_A antibody at the concentration described above. They were then incubated for 1 h with biotinylated antirabbit IgG (Vector Laboratories, Inc.) as the secondary antibody according to the concentration described in the instruction. The sections were then incubated with the ABC reagent for 30 min, and positive reactions were visualized by incubation with the peroxidase substrate solution containing 3,3'-diaminobenzidine tetrahydrochloride. Nuclei were counterstained with Mayer’s hematoxylin.

To quantify the number of ß_A-subunit-positive cells, the numbers of ß_A-positive cells and total nuclei were counted in 30 fields per section at the magnification of x400. Five sections were counted per mouse. The ratio of ß_A-positive cells/total cells was calculated. To quantify the number of islet-like cell cluster (ICC), the number of ICC was counted in 30 fields per section and 30 sections were counted per mouse.

Measurement of plasma glucose and insulin concentrations
Blood was collected in heparinized hematocrit tubes at different time points after STZ injection and partial pancreatectomy. The plasma glucose concentration was measured with a glucose analyzer (Sanwa Chemical Co., Nagoya, Japan), and the insulin concentration was measured by a time-resolved immunofluorometric assay system as described previously (24). All samples were assayed in duplicate.

Statistical analysis
Statistical analysis was done by one-way ANOVA. For comparison between the two groups, the unpaired Student’s t test was used. The P value less than 5% was considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of activin subunits and the type II activin receptor in the pancreas
To examine whether or not subunits of activin were expressed in the pancreatic duct, we isolated ducts from the pancreas and examined their mRNA expression by RT-PCR. As shown in Fig. 1, both ß_A- and ß_B-subunits of activin were expressed in the pancreatic ducts. The type II and type IIB activin receptors were also expressed in the duct. Note that the expression of insulin and amylase was negligible in our duct preparation. Expression of ß_A- and ß_B-subunits of activin in the pancreas was also studied by immunohistochemistry. Immunoreactivity of ß_A was detected in the islets as well as in the ducts. We stained a consecutive section with polyclonal anti-CK antibody, a marker of ductal cells, and found that immunoreactivity of CK was colocalized with immunoreactive ß_A. Both ß_A and CK were colocalized in small ductules and many of the intercalated ducts (Fig. 2A). Immunoreactive ß_A was also observed in the center of pancreatic acini, suggesting that some of centroacinar cells were positive for the ß_A-subunit (Fig. 2B). We then studied whether or not the ß_A-subunit was colocalized with nestin in the pancreas. There were some nestin-positive cells in the islet (Fig. 2C). Nestin-positive cells were also found in the duct epithelium and in periductal area (Fig. 2C). The expression of nestin in intercalated ducts or centroacinar cells was low. Some of the ductal cells expressed both the ß_A-subunit and nestin (Fig. 2D). We also studied the expression of the ß_B-subunit of activin. Immunoreactivity of the ß_B-subunit was observed in the pancreatic duct (Fig. 2E). Immunoreactive ß_B was found in large intralobular ducts but rarely in small ductules or intercalated ducts. We next studied the expression of the type II activin receptor. Immunoreactivity of the type II receptor of activin was detected in the islets (Fig. 3A). The type II receptor was detected in ß-cells and some of cells. However, immunoreactivity of the type II receptor was not detected in the ductal cells (data not shown). In contrast, the expression of the type IIB activin receptor was found in the pancreatic duct. It is found in large ducts and some of ductules (Fig. 3B). The type IIB receptor was also expressed in blood vessels, identified by the expression of factor VIII (Fig. 3B). Immunoreactivity of the type IIB receptor was not detected in islets. Expression of the type IIB activin receptor was observed in some of the nestin-positive cells in the pancreatic duct (Fig. 3C). Results of the expression of activin subunits and type II receptors are summarized in Table 1.

View larger version (34K): [in this window] [in a new window]   Figure 1. Expression of mRNA for ßA- and ßB-subunits of activin, type II and type IIB activin receptors, amylase and insulin in the pancreatic ducts. RNA was extracted from isolated pancreatic ducts and Islets. The expression of mRNA for ßA- and ßB-subunits, type II and type IIB activin receptors, amylase, and insulin was measured by RT-PCR.

View larger version (60K): [in this window] [in a new window]   Figure 2. Expression of the ßA- and ßB-subunits of activin in the pancreas. A, Consecutive sections were stained with anti-ßA (left) and anti-CK (right) antibodies. Magnification, x200. B, A section was immunostained with anti-ßA antibody (brown) and counterstained with hematoxylin. ßA-Subunit was observed in the center of some acini. Magnification, x1000. C, Consecutive sections were double stained with antiinsulin (red) and either anti-ßA (green) or antinestin (green) antibodies. Magnification, x200. Nestin-positive cells were observed in the islet and the duct. There were some nestin-positive cells in periductal area. D, Consecutive sections were stained with anti-ßA (left) and antinestin (right) antibodies. Magnification, x400. ßA-Positive ductal cells coexpressed nestin. E, A section was double-stained with anti-ßB (red) and anti-CK (green) antibodies. Magnification, x400.

View larger version (34K): [in this window] [in a new window]   Figure 3. Expression of the type II and type IIB activin receptors in the pancreas. A, Pancreatic sections were double-stained with antibodies against type II activin receptor (green) and either insulin (ins, red) or glucagon (glu, red). The type II receptor was detected in ß-cells and some of -cells. B, A section was double stained with anti-type IIB activin receptor (right) and antifactor VIII (left) antibodies. The type IIB receptor was expressed in the duct and in factor VIII-positive endothelial cells (arrowheads). Magnification, x400. C, A section was double-stained with antinestin (red) and antitype IIB activin receptor (green) antibodies. The type IIB receptor was expressed in duct epithelia, some of which coexpressed nestin.

View larger version (43K): [in this window] [in a new window]   Figure 4. Changes in the expression of the ßA- and ßB-subunits in the pancreatic ducts in STZ-treated mice. A, STZ (200 µg/g) was injected and the pancreatic ducts were isolated at various time points. RNA was extracted and the mRNA for ßA, ßB, and GAPDH was measured by real-time RT-PCR. Ratios of ßA/GAPDH and ßB/GAPDH are shown. Values are the mean ± SE for six experiments. , ßA; , ßB. B, Mice were injected with vehicle or STZ (200 µg/g) and pancreatic sections obtained from vehicle- (control) and STZ-treated (STZ) mice after 24 h were immunostained with anti-ßA and anti-CK antibodies. Magnification, x400. C, Pancreatic sections obtained from control (left) and STZ-treated (right) mice 24 h after the injection was stained with anti-ßA (green) and antiinsulin (red) antibodies. Magnification, x400.

View larger version (30K): [in this window] [in a new window]   Figure 5. Changes in the expression of the ßA- and ßB- subunits in the pancreatic ducts in remnant pancreas after partial pancreatectomy. Pancreatic ducts were isolated from remnant pancreas at various time points after partial pancreatectomy. RNA was extracted and mRNA for ßA, ßB, and GAPDH was measured by real-time RT-PCR. Ratios of ßA/GAPDH and ßB/GAPDH are shown. Values are the mean ± SE for six experiments. , ßA; , ß,ßB.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present results indicate that activins are expressed in non-ß-cells of the islets and in ductal cells in mice. We previously reported that immunoreactivity of the ß_A-subunit was localized in the secretory granules of and cells of rat pancreatic islets (19). Others reported that immunoreactive ß_A was localized in ß-cells of rat islets (26). These differences are due at least partly to the complex feature of antigenicity of the ß_A-subunit. For example, Ogawa et al. (27) prepared two anti-ß_A antibodies raised against synthetic polypeptides corresponding to two different portions of ß_A-subunits. Surprisingly, when these two antibodies were used for immunohistochemistry, one antibody stained ß-cells while the other stained non-ß-cells of rat islets. It is still difficult at present to definitely identify the type of islet cells expressing activin. Nevertheless, all of the previous studies (11, 26, 27) agree that immunoreactive ß_A was not detected in ductal cells of the rat pancreas. When measured by RT-PCR, however, we detected mRNA for the ß_A- and ß_B-subunits in isolated rat pancreatic ducts (unpublished data). Again, the inability to detect these subunits by immunohistochemistry may have been due to inadequate presentation of the epitope in rat ductal cells. In any event, both ß_A- and ß_B-subunits are expressed in pancreatic ducts in adult mice, which was confirmed by RT-PCR and immunohistochemistry.

Although the mechanisms by which STZ and partial pancreatectomy reduce the functional ß-cell mass differ considerably, the expression of activins was up-regulated by these two procedures. Newly formed insulin-producing cells are often observed in duct epithelial cells after the destruction of ß-cells (16, 17, 18). Presumably, progenitor cells exist in or by the duct (28) and convert to insulin-producing cells in these conditions. Alternately, mature duct epithelial cells, or possibly acinar cells, convert to dedifferentiated cells, which then differentiate to insulin-producing cells (29). In this regard, Zulewski et al. (28) showed recently that nestin-positive cells existed in and by the pancreatic duct. They also showed that the nestin-positive cells derived from cultured islets converted to insulin-producing cells. They postulate that the nestin-positive cells represent the multipotent pancreatic stem cells. As shown in Fig. 2D, some of the nestin-positive cells expressed the ß_A-subunit. Hence, as in the progenitor cells in embryonic pancreas, at least some of the pancreatic progenitor cells may express the ß_A-subunit of activin. Previous studies have shown that activins play critical roles in the formation of pancreatic endocrine cells: activin or related molecules controls differentiation of progenitors to endocrine cells in fetal pancreas (10); activin A converts amphicrine AR42J cells into pancreatic polypeptide-producing endocrine cells (24, 30); activin A induces differentiation of fetal pancreatic cells (31) and the nestin-positive progenitor cells in rat pancreatic islets (28); and furthermore, activin A induces the expression of neurogenin 3 (23), which is critical in the differentiation of pancreatic endocrine cells (32, 33, 34). Consequently, it is quite likely that activins induced in ductal cells may play a role in initiating differentiation of progenitors in the pancreatic duct into insulin-producing cells.

Reduction of the ß-cell mass either by STZ treatment or partial pancreatectomy augmented the expression of activin subunits in the duct. What is the mechanism responsible for this up-regulation? As mentioned above, STZ and pancreatectomy reduce functional ß-cell mass by quite different mechanisms with different time courses. Nevertheless, both procedures induce similar patterns of changes in the expression of activins. There must have been a common trigger to stimulate the expression. In this regard, two procedures induced similar changes in glucose metabolism. The plasma glucose concentration increased rapidly in STZ treated and partially pancreatectomized mice. Because glucose infusion alone rapidly initiates ß-cell neogenesis (35), it is possible that a transient increase in plasma glucose triggers the induction of ß-subunits. Nonetheless, elevation of plasma glucose induced by ip administration of glucose did not augment the expression of ß-subunits. Elevation of glucose may not be a trigger of induction of activins in the ducts. There may be other factors regulating the expression of activin subunits. Inflammatory cytokines, for example, are possible candidates inducing the expression of the ß-subunits. In other type of cells, mRNA for the ß_A-subunit of activin is induced by cAMP and phorbol ester, and a combination of these two agents synergistically up-regulated the expression of ß_A (36). Consistent with this observation, the cAMP-responsive element and AP-1 binding site are found in the regulatory region of the ß_A gene (37). The 5'-flanking region of the ß_B gene contains cAMP-responsive element and multiple Simian virus 40 promoter region binding factor 1 (Sp-1) binding sites (38, 39). Yet, the molecular mechanism regulating the transcription of ß_A and ß_B genes has not been investigated completely, and further studies are clearly necessary to elucidate the regulation of transcription of activins in the pancreatic duct. In any event, little is known about the initial event of the ß-cell neogenesis. Elucidation of the initial step of the ß-cell neogenesis does not only help to understand the regulatory mechanism to maintain the normal ß-cell mass but also provides a potential therapeutic approach to promote ß-cell neogenesis. In this regard, induction of activins in the pancreatic duct, where ß-cell neogenesis is initiated, may be one of the key events in the initiation of ß-cell neogenesis.

	Acknowledgments

 
The authors are grateful to Mayumi Odagiri for her technical and secretarial assistance.

	Footnotes

 
This work was supported by a Grant-in Aid from the Ministry of Science, Education, Sports and Culture of Japan and grants from the Naito Foundation, the Inamori Foundation, the Suzuken Memorial Foundation and the Ichiro Kanehara Foundation.

Abbreviations: ABC, Avidin-biotin complex; CK, cytokeratin; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde phosphate dehydrogenase; ICC, islet-like cell cluster; STZ, streptozotocin.

Received January 24, 2002.

Accepted for publication May 7, 2002.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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