猪胰腺干细胞分离鉴定
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摘要
糖尿病是全球性医学难题,目前,对糖尿病的治疗虽然有很多方法,但都不能从根本上解决问题。主要受制于供体不足和免疫排斥的问题。因此,人们寄希望于干细胞替代疗法。干细胞增殖力强、数量充足、移植排斥反应相对较低,更重要的是胰腺干细胞可塑性强,可以分化形成胰腺组织的各种细胞。猪胰腺干细胞的研究,目的在于解决干细胞来源的问题.
本人在实验室以往的工作基础上,进一步分离、鉴定猪胰腺干细胞。无菌采集胎猪胰腺120例,采用酶消化法,用RPMI1640加入1mmol/L丙酮酸钠,71.5μmol/Lβ-巯基乙醇,20ng/mL EGF、20ng/mL bFGF,100U/mL青霉素100U/mL、链霉素0.1mg/mL,添加10% FBS作培养基培养细胞。结果有59例仅获得原代细胞,部分传至6～8代后,细胞大量飘浮死亡。同时观察到细胞传至6～8代时,随着细胞的大量死亡漂浮,皿底始终零散地保留一些圆形的、折光性强的小细胞。此时采用含20%血清的上述培养液后,皿中的小细胞在换液24h后形态发生变化,变大、变长,迅速扩增。48h后,每个小圆细胞形成一个克隆,96h后形成大的克隆,直径超过100um,随后细胞不断从集落边缘迁徙出来,细胞间相互连接,形成网络状生长,5～6天就会融汇成单层,此类细胞可以多次进行传代。由此发现胰腺干细胞不能传过7～8代的制约因素,成功地突破了胰腺干细胞扩增难题,并且一次获得4株胰腺干细胞,其中一株已经顺利传至64代,另3例传至10代,各代均有冻存;在诱导分化实验中观察到如果用无血清的培养基诱导胰腺干细胞向β细胞分化,细胞可以大量形成胰岛细胞团,但是成团后细胞活力很快下降,发黑、漂浮甚至死亡。因此,进一步探索了诱导条件,采用含血清的诱导液诱导,不仅可以促使胰腺干细胞向胰岛内分泌细胞分化,而且有效地避免了细胞分化后活力下降的问题。利用高糖刺激胰岛素释放实验,胰岛素和c-肽的分泌量分别可以达到306.87±20.72 mIU/mL和0.31±0.11ng/mL。
经免疫组化检测,细胞表达目前公认的胰腺干细胞特异性标记物,如胰肠同源域因子1、葡萄糖转运子2、巢蛋白和波形蛋白,经RT-PCR检测,细胞表达胰肠同源域因子1、葡萄糖转运子2、胰淀素前体、胰岛素、生长抑素,确定分离获得的细胞为胰腺干细胞。细胞经成骨细胞诱导液诱导后,有明显的细胞聚集成团块状物的形成,经茜素红染色呈阳性;向神经细胞诱导后,细胞NF-200染色阳性;细胞亦表达胚胎干细胞的一些标志,如强表达胚胎干细胞的表面标志物OCT-4、胚胎阶段特异性抗原(1Stage-specific embryonic antigens SSEA-1);用流式细胞仪证实细胞表达骨髓间充质干细胞的一些表面标志,如CD29,CD44,CD166。通过裸鼠致瘤性实验证实所分离的胰腺干细胞无致瘤性。并制备了大鼠糖尿病模型,将诱导后的细胞经腹腔注射给大鼠模型用于治疗,仅在注射后的2～4天内,血糖略有下降。
Diabetes mellitus is a devastating disease all over the world. With the increasing morbidity of diabetes mellitus,the diabetes mellitus has now become the third highest incidence following cardiovascular disease and tumour which are chronic diseases heavily threatening people’s common health.Its syndrome caused by hyperglycosemia put heavy burden on patient not only in economic but also in spirit.
Diabetes mellitus has a increase trend.In our country,its incidnce in 1994 asend to 3 times to 1980.and it was estimated that diabetes will increse to 1 billion in following 30 years and its incidence will be 5% to 10% of the total number of people.Thus, it is urgent studing on prevention and treatment methods for Diabetes mellitus.
The general principle of diabetes therapy is to supplied with insulin rapidly, availably and chronically to replenish the self-secreting lack of insulin.Restablishing a endogenic insulin secret system is the best way to fufil these demand.The pancreatic stem cells can be unlimitly proliferated and differantiated into pancreatic endocrine cells.if we get this multipotential cells easily and directed its differentiation according to patient’s need.that should give hope to diabetes therapy.
Following former researcher methods,pancreas collected from 90-day old porcine fetus was dissected and then digested with 0.25% trypsin for 10 min. The primary cells were cultured in medium of RPMI 1640 with 10%FBS (added 1mmol/L pyravate, 71.5μmol/Lβ-Me, 20ng/ml EGF,20ng/ml bFGF).the cell derived from porcine fetus were identified as pancreatic stem cell by immunochemical method.During cells cultivation and passage,we have found that the pancreatic stem cell couldn’t be passed over 8 time only in 10% serum concentration. Low concertration of serum made most of the cells died, only a few cells survived that have the small and round morphology attached to the surface of culture plate.But we found if we changed medium to RPMI 1640 with 20% FBS (supplement with 1 mM pyravate, 71.5μMβ-Me, 20 ng/ml EGF, 20 ng/ml bFGF) at this time ,these little cells started to become bigger and proliferate to form clusters. It grew to confluence in another 6 days. After the first few passages, cell morphology was diversified into mesenchymal-like and fibroblast-like cells. Four cell strains were finally purified by this method. One of the strains was continually grown for 64 passages in 14-month culture period and presented the immortalized feature. The results of immunochemistry study showed that the isolated cell expressed pancreatic duodenal homeobox-1 (PDX-1), glucose transporter-2 (GLUT-2) , Proliferating cell nuclear antigen (PCNA),nestin and vimentin biomarkers that were thought as markers of the pancreatic stem cell.
To induce cell differentiation,cells grown in medium of DMEM/F12 with 2% B27、0.5%BSA、10mM nicotinamide,5ng/ml HGF,10ng/ml EGF,20nM GLP-1,100U/ml Penicillin/Streptomycin Solution for 6-8 days were formed the islet-like cell clusters) The results of immunochemistry study showed that the isolated cell retained PDX-1,GLUT-2 and vimentin biomarkers when cells forming the cluster. After differentiation, several endocrine hormones that were specially produced from pancreatic islet cells were also detected in the cell that included insulin, glucagon, somatostatin and polypeptide. This investigation revealed that the cell was the pancreatic stem cell and was able to differentiate to all four kinds of pancreatic islet like cells in vitro.we also found the viability of formed ICCs were not good in induction medium without serum,so we added serum in induction medium,the results revealed that the pancreatic stem cells can also differentiated into endocrine cells in induction medium with serum and retain good viability. By means of radioimmunoassay(RIA),βcells induced from the pancretic stem cell had an increase in secretion of insulin and c-peptide. The singleβcelI was also brightred for dithizone(DTZ) staining.These results indicated that there are multipotent stem cells in pancreas. The present study offers an approach to isolate and expand pancreatic stem cells from porcine fetus, and differentiate them into functionalβcells in vitro. It likely become an new source for diabetes therapy through xenotransplantation of pancreatic stem cells.
The immunohistochemical staining also showed that the isolated cells express the markers of the embryonic stem cell including SSEA-1 and Oct4.at the same time,cells were positive for AKP, which indicated that the cells isolated possess the multipotential properties. Prior to treating with medium supplemented 5% NBS and 1 mmol/Lβ-mercaptoethanol(β-Me) for 24h, the isolated cells were treated with 5 mmol/Lβ-Me, no serum for 4h sequentia to differentiate toward neural cells. The induced cells expressed NF,the marker of neural cells.
Differentiation of isolated cells toward the osteoblast lineage can be induced by supplementing 0.1μmol/L dexamethasone, 10 mmol/Lβ-glycerophosphate and 50μg/mL ascorbic acid. The cells were induced forming cluster after 7 day’s culture.The cells were positive stained with alkaline phosphatase (AKP).
Cells were injected into groin of nude mouse at 106,tumour were not found that imply the pancreatic stem cell has no oncogenesis。We injectedβcells induced from the pancretic stem cell into cavum abdominis of rat which were diabets modle treated by STZ(streptocizone),hyperglycemia of rat were not be remited during the period of 60 days treatment.that imply rejection reaction was the obstacle for apply the pancretic stem cell to therapy,its effect should be satisfied if its rejection reaction were decresed.
In conclusion, four individual pancreatic stem cells were isolated from porcine pancreas tissue in which one cell strain was continually cultured for 14 months (to 64 passages). This study provided a simplified procedure to isolate and culture pancreatic stem cells from animal pancreas, and established an animal cell strain for cell therapy in human diabetic mellitus.
本人在实验室以往的工作基础上,进一步分离、鉴定猪胰腺干细胞。无菌采集胎猪胰腺120例,采用酶消化法,用RPMI1640加入1mmol/L丙酮酸钠,71.5μmol/Lβ-巯基乙醇,20ng/mL EGF、20ng/mL bFGF,100U/mL青霉素100U/mL、链霉素0.1mg/mL,添加10% FBS作培养基培养细胞。结果有59例仅获得原代细胞,部分传至6～8代后,细胞大量飘浮死亡。同时观察到细胞传至6～8代时,随着细胞的大量死亡漂浮,皿底始终零散地保留一些圆形的、折光性强的小细胞。此时采用含20%血清的上述培养液后,皿中的小细胞在换液24h后形态发生变化,变大、变长,迅速扩增。48h后,每个小圆细胞形成一个克隆,96h后形成大的克隆,直径超过100um,随后细胞不断从集落边缘迁徙出来,细胞间相互连接,形成网络状生长,5～6天就会融汇成单层,此类细胞可以多次进行传代。由此发现胰腺干细胞不能传过7～8代的制约因素,成功地突破了胰腺干细胞扩增难题,并且一次获得4株胰腺干细胞,其中一株已经顺利传至64代,另3例传至10代,各代均有冻存;在诱导分化实验中观察到如果用无血清的培养基诱导胰腺干细胞向β细胞分化,细胞可以大量形成胰岛细胞团,但是成团后细胞活力很快下降,发黑、漂浮甚至死亡。因此,进一步探索了诱导条件,采用含血清的诱导液诱导,不仅可以促使胰腺干细胞向胰岛内分泌细胞分化,而且有效地避免了细胞分化后活力下降的问题。利用高糖刺激胰岛素释放实验,胰岛素和c-肽的分泌量分别可以达到306.87±20.72 mIU/mL和0.31±0.11ng/mL。
经免疫组化检测,细胞表达目前公认的胰腺干细胞特异性标记物,如胰肠同源域因子1、葡萄糖转运子2、巢蛋白和波形蛋白,经RT-PCR检测,细胞表达胰肠同源域因子1、葡萄糖转运子2、胰淀素前体、胰岛素、生长抑素,确定分离获得的细胞为胰腺干细胞。细胞经成骨细胞诱导液诱导后,有明显的细胞聚集成团块状物的形成,经茜素红染色呈阳性;向神经细胞诱导后,细胞NF-200染色阳性;细胞亦表达胚胎干细胞的一些标志,如强表达胚胎干细胞的表面标志物OCT-4、胚胎阶段特异性抗原(1Stage-specific embryonic antigens SSEA-1);用流式细胞仪证实细胞表达骨髓间充质干细胞的一些表面标志,如CD29,CD44,CD166。通过裸鼠致瘤性实验证实所分离的胰腺干细胞无致瘤性。并制备了大鼠糖尿病模型,将诱导后的细胞经腹腔注射给大鼠模型用于治疗,仅在注射后的2～4天内,血糖略有下降。
Diabetes mellitus is a devastating disease all over the world. With the increasing morbidity of diabetes mellitus,the diabetes mellitus has now become the third highest incidence following cardiovascular disease and tumour which are chronic diseases heavily threatening people’s common health.Its syndrome caused by hyperglycosemia put heavy burden on patient not only in economic but also in spirit.
Diabetes mellitus has a increase trend.In our country,its incidnce in 1994 asend to 3 times to 1980.and it was estimated that diabetes will increse to 1 billion in following 30 years and its incidence will be 5% to 10% of the total number of people.Thus, it is urgent studing on prevention and treatment methods for Diabetes mellitus.
The general principle of diabetes therapy is to supplied with insulin rapidly, availably and chronically to replenish the self-secreting lack of insulin.Restablishing a endogenic insulin secret system is the best way to fufil these demand.The pancreatic stem cells can be unlimitly proliferated and differantiated into pancreatic endocrine cells.if we get this multipotential cells easily and directed its differentiation according to patient’s need.that should give hope to diabetes therapy.
Following former researcher methods,pancreas collected from 90-day old porcine fetus was dissected and then digested with 0.25% trypsin for 10 min. The primary cells were cultured in medium of RPMI 1640 with 10%FBS (added 1mmol/L pyravate, 71.5μmol/Lβ-Me, 20ng/ml EGF,20ng/ml bFGF).the cell derived from porcine fetus were identified as pancreatic stem cell by immunochemical method.During cells cultivation and passage,we have found that the pancreatic stem cell couldn’t be passed over 8 time only in 10% serum concentration. Low concertration of serum made most of the cells died, only a few cells survived that have the small and round morphology attached to the surface of culture plate.But we found if we changed medium to RPMI 1640 with 20% FBS (supplement with 1 mM pyravate, 71.5μMβ-Me, 20 ng/ml EGF, 20 ng/ml bFGF) at this time ,these little cells started to become bigger and proliferate to form clusters. It grew to confluence in another 6 days. After the first few passages, cell morphology was diversified into mesenchymal-like and fibroblast-like cells. Four cell strains were finally purified by this method. One of the strains was continually grown for 64 passages in 14-month culture period and presented the immortalized feature. The results of immunochemistry study showed that the isolated cell expressed pancreatic duodenal homeobox-1 (PDX-1), glucose transporter-2 (GLUT-2) , Proliferating cell nuclear antigen (PCNA),nestin and vimentin biomarkers that were thought as markers of the pancreatic stem cell.
To induce cell differentiation,cells grown in medium of DMEM/F12 with 2% B27、0.5%BSA、10mM nicotinamide,5ng/ml HGF,10ng/ml EGF,20nM GLP-1,100U/ml Penicillin/Streptomycin Solution for 6-8 days were formed the islet-like cell clusters) The results of immunochemistry study showed that the isolated cell retained PDX-1,GLUT-2 and vimentin biomarkers when cells forming the cluster. After differentiation, several endocrine hormones that were specially produced from pancreatic islet cells were also detected in the cell that included insulin, glucagon, somatostatin and polypeptide. This investigation revealed that the cell was the pancreatic stem cell and was able to differentiate to all four kinds of pancreatic islet like cells in vitro.we also found the viability of formed ICCs were not good in induction medium without serum,so we added serum in induction medium,the results revealed that the pancreatic stem cells can also differentiated into endocrine cells in induction medium with serum and retain good viability. By means of radioimmunoassay(RIA),βcells induced from the pancretic stem cell had an increase in secretion of insulin and c-peptide. The singleβcelI was also brightred for dithizone(DTZ) staining.These results indicated that there are multipotent stem cells in pancreas. The present study offers an approach to isolate and expand pancreatic stem cells from porcine fetus, and differentiate them into functionalβcells in vitro. It likely become an new source for diabetes therapy through xenotransplantation of pancreatic stem cells.
The immunohistochemical staining also showed that the isolated cells express the markers of the embryonic stem cell including SSEA-1 and Oct4.at the same time,cells were positive for AKP, which indicated that the cells isolated possess the multipotential properties. Prior to treating with medium supplemented 5% NBS and 1 mmol/Lβ-mercaptoethanol(β-Me) for 24h, the isolated cells were treated with 5 mmol/Lβ-Me, no serum for 4h sequentia to differentiate toward neural cells. The induced cells expressed NF,the marker of neural cells.
Differentiation of isolated cells toward the osteoblast lineage can be induced by supplementing 0.1μmol/L dexamethasone, 10 mmol/Lβ-glycerophosphate and 50μg/mL ascorbic acid. The cells were induced forming cluster after 7 day’s culture.The cells were positive stained with alkaline phosphatase (AKP).
Cells were injected into groin of nude mouse at 106,tumour were not found that imply the pancreatic stem cell has no oncogenesis。We injectedβcells induced from the pancretic stem cell into cavum abdominis of rat which were diabets modle treated by STZ(streptocizone),hyperglycemia of rat were not be remited during the period of 60 days treatment.that imply rejection reaction was the obstacle for apply the pancretic stem cell to therapy,its effect should be satisfied if its rejection reaction were decresed.
In conclusion, four individual pancreatic stem cells were isolated from porcine pancreas tissue in which one cell strain was continually cultured for 14 months (to 64 passages). This study provided a simplified procedure to isolate and culture pancreatic stem cells from animal pancreas, and established an animal cell strain for cell therapy in human diabetic mellitus.
引文
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