TNFα对人胚胎胰岛分泌IL-6及对胰岛分泌功能的影响

为了探讨ｒｈＴＮＦα对人胚胎胰岛分泌ＩＬ６以及对胰岛分泌功能的影响，观察了ｒｈＴＮＦα对原代培养的人胚胎胰岛细胞的作用特点。结果表明：①经ｒｈＴＮＦα刺激后胰岛培养上清ＩＬ６含量显著升高，在一定范围内呈剂量依赖关系；②斑点杂交结果显示ｒｈＴＮＦα刺激的胰岛ＲＮＡ中ＩＬ６ｍＲＮＡ含量高于对照组；③人胚胎胰岛受ｒｈＴＮＦα刺激后培养上清胰岛素含量降低；④人胚胎胰岛受ｒｈＴＮＦα刺激后培养上清胰高血糖素分泌能力明显升高，分泌趋势与ＩＬ６相似。

LPS对人胚胎胰岛分泌IL6的调控效应

目的：为了探讨免疫介质ＬＰＳ对胚胎胰岛分泌ＩＬ６以及对胰岛功能的影响，以便为研究免疫系统与内分泌系统的内在联系提供依据。方法：采用常规消化方法分离人胚胎胰岛并对其进行原代培养，加入浓度为１０ｍｇ／ＬＬＰＳ，经不同时相收获上清，测定ＩＬ６、胰岛素、胰高血糖素含量和ＩＬ６ＭｃＡｂ中和试验，对ＬＰＳ刺激和对照组胰岛ＲＮＡ进行ＩＬ６ＲＮＡ斑点杂交。结果：①人胚胎胰岛经ＬＰＳ刺激分泌ＩＬ６的能力明显加强，在体外培养８ｄ，ＩＬ６含量达高峰，为对照组的１４５倍；②人胚胎胰岛在换新鲜培养液后用ＬＰＳ二次刺激，仍可继续分泌高效价的ＩＬ６，高峰时相为对照组的２５倍；③ＩＬ６ＭｃＡｂ对胰岛培养上清中ＩＬ６有很强的中和效应；④斑点杂交结果显示ＬＰＳ刺激后胰岛ＲＮＡ中ＩＬ６ｍＲＮＡ含量明显高于对照组；⑤人胚胎胰岛受ＬＰＳ刺激后胰岛素分泌能力加强，胰高血糖素分泌能力明显降低。

提高冻存胚胎胰岛移植效果的实验研究

目的探讨三维组织细胞旋转培养系统(RCCS)对胚胎胰岛冻存复苏后质量的影响。方法将胚胎胰岛平均分为3组,实验组1、2为胚胎胰岛冻存前后分别用RCCS培养和普通培养,对照组新鲜胰岛经RCCS培养。切取胚胎胰腺,胶原酶V消化,纯化。然后进行标准冻存步骤,复苏后继续培养。并检测各组胰岛数量、活性、胰岛素刺激实验结果。结果纯化后收获胰岛最多每个胚胎5012.73IEQ,最少2432.68IEQ,平均(3548.07±273.46)IEQ。微重力培养组胰岛细胞存活率、胰岛素释放量、胰岛素刺激指数等均高于普通培养组。移植经过微重力培养的(2000±1)%IEQ新鲜胚胎胰岛或冻存胚胎胰岛在移植后1周内可达100%纠正糖尿病。结论微重力旋转培养有利于胰岛细胞的生长繁殖,使胰岛具有更好的胰岛素分泌能力,该方法同胰岛冻存相结合,可以进一步提高胰岛的冻存效果,为胰岛库的成功建立探索出一条新的途径。

从宫内发育研究代谢综合征的发病机制

宫内发育迟缓与成年后代谢综合征(metabolic syndrome,MS)关系密切。研究发现,宫内营养不良可通过影响胎儿胰岛β细胞的发育和功能、干扰胎儿的糖脂代谢、调节激素水平、调控基因的修饰与表达等多种途径影响胎儿的生长发育与物质代谢,对生后MS的发病起着重要的推动作用。

炎性细胞因子对人胚胎胰岛功能及其分泌IL_6的调控效应

目的 探讨正常人胚胎胰岛在 Ｉ Ｌ１β、 Ｔ Ｎ Ｆα、 Ｉ Ｆ Ｎγ作用下， Ｉ Ｌ６ 的分泌及基因表达状况。方法 采用常规消化方法分离人胚胎胰岛并进行培养，分别加入ｒｈ Ｉ Ｌ１β、ｒｈ Ｔ Ｎ Ｆα、ｒｈ Ｉ Ｆ Ｎγ或同时加入２ 种或３ 种细胞因子，作用２４ 、４８ 小时收上清，进行 Ｉ Ｌ６ 、胰岛素、胰高血糖素活性检测和 Ｉ Ｌ６单抗中和实验，同时对３ 种细胞因子联合刺激的胰岛 Ｒ Ｎ Ａ 进行 Ｉ Ｌ６ ｍ Ｒ Ｎ Ａ 斑点杂交。结果 ３ 种细胞因子单独或联合刺激均可大幅度提高 Ｉ Ｌ６ 和胰高血糖素的分泌，同时不同程度地抑制胰岛素的分泌，细胞因子刺激胰岛的时间和强度与 Ｉ Ｌ６ 、胰岛素、胰高血糖素的分泌有明显的关系。斑点杂交结果显示３ 种细胞因子联合刺激的胰岛 Ｒ Ｎ Ａ 中 Ｉ Ｌ６ ｍ Ｒ Ｎ Ａ 含量明显高于对照组。结论 炎性细胞因子调节胰岛分泌 Ｉ Ｌ６ 和 Ｉ Ｌ６ 基因表达。

Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

AIM： To enhance the differentiation of insulin producing cell （IPC） ability from embryonic stem （ES） cells in vitro. METHODS： Four-day embryoid body （EB）-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of NucleofectorTM electroporator （Amaxa biosystems, Germany） in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectincoated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination.The differentiation status of these cells was monitored by semi-quantitative PCR （SQ-PCR） detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixll, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry （IHC）, and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease （SCID） pretreated with streptozotocin （STZ） were used to eliminate plasma glucose restoration after pax4^＋ ES implantation. RESULTS： A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixll, pdxl, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION： Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasrnid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, the potential of pax-4-nucleofected cells in medical treatment is promising.

Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer

AIM： To investigate the ability of a genetically altered embryonic stem （ES） cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene.METHODS： Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body （EB） culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone （DTZ）, a zincchelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells.The outgrowths were incubated in DTZ solution （final concentration, 100μg/mL） for 15 rain before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA.RESULTS： DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells （Nkx-ES cells）, which was as much as 2 wk earlier, than those in the culture of parental ES cells （wt-ES）. The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 （PDX1）, proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters.Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated.CONCLUSION： Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.

In vitro derivation of functional insulin-producing cells from human embryonic stem cells

The capacity for self-renewal and differentiation of human embryonic stem （ES） cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid （RA） was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdxl and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin （STZ）-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdxl, glucokinase, nkx6.1, lAPP, pax6 and Tcfl. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.

Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been made in generating insulin-secreting 13 cells from pluripotent mouse embryonic stem cells （ESCs）.The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions. Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of 13 cells such as insulinl, insulin2, Isletl, MafA, insulinoma-associated antigen 1 （IA1） and so on, indicating a similar gene expression pattern to adult islet 13 cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion, this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro, but also leaded to the development of new strategies to derive transplantable islet-replacement 13 cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.

白细胞介素6个对人胚胎胰岛分泌胰岛素的促进效应

我们采用胶原酶Ｖ（０．５ｍｇ／ｍｌ）逐级消化方法获取胚胎胰岛细胞，对其进行原代培养并加入不同剂量的白细胞介素６（ＩＬ－６）。结果表明胚胎胰岛在培养２４小时后，ＩＬ－６可以促进胰岛素的分泌，与对照组比较差异非常显著（ｔ＝３１．９７，Ｐ〈０．００１）；结果还表明ＩＬ－６促进胰岛素的分泌呈剂量依赖关系，提示ＩＬ－６在人胰腺代谢中可能起重要作用。

Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media： distinct requirements for TGF-β and IGF-Ⅱ

A paracrine regulation was recently proposed in human embryonic stem cells （hESCs） grown in mouse embryonic fibroblast （MEF）-conditioned media （MEF-CM）, where hESCs spontaneously differentiate into autologous fibroblastlike cells to maintain culture homeostasis by producing TGF-β and insulin-like growth factor-lI （IGF-Ⅱ） in response to basic fibroblast growth factor （bFGF）. Although the importance of TGF-β family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-Ⅱ. In order to ease hESC cul- ture conditions and to reduce xenogenic components, we sought （i） to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts （HFFs） and human mesenchymal stem cells （hM- SCs） rather than MEF-CM, and （ii） to analyze whether the cooperation of bFGF with TGF-β and IGF-Ⅱ to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell （MSC）-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors （FGFR1-4） and specifically produce TGF-β in response to bFGF. However, HFFs but not MSCs secrete IGF-Ⅱ. Despite the absence of IGF-Ⅱ in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-Ⅱ may be dispensable for hESC pluripotency. In fact, IGF-Ⅱ blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-Ⅱ-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-β and IGF-Ⅱ in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

Development of a Synthetic Medium for the in Vitro Culture of Bovine Embryos

The aim of this study was to develop a synthetic medium for the in vitro culture of bovine embryos, using various growth factors and cytokines （GF-CYK）： insulin-like growth factorl （IGF-Ⅰ）, insulin-like growth factorⅡ （IGF-Ⅱ）, basic fibroblast growth factor （bFGF）, leukemia inhibitory factor （LIF）, granulocyte-macrophage colony stimulating factor （GM-CSF） and transforming growth factor beta Ⅰ （TGF-β1） ＋ hyaluronan （HA） ＋ recombinant albumin （RA）. The embryos were cultured in synthetic oviduct fluid （SOF） supplemented with： treatment 1 （T1）： bovine serum albumin （BSA） ＋ insulin, transferrin and selenium （ITS） （control）; or treatment 2 （T2）： GF-CYK ＋ HA ＋ RA. The blastocyst rates were not significantly different between TI and T2, at seven days post fertilization （dpf） （28.9% ± 2.4% and 31.8% ±2.2%）, and at 8 dpf （36.5% ±2.4% and 39.1% ±1.9%）, respectively （P 〉 0.05）. The total cell number （TCN） was significantly higher with T2 than that with T1 at 7 dpf（164.9 ±5.3 and 149.7 ±4.0） and 8 dpf （182.7 ±6.4 and 165.0 ±5.5） （P 〈 0.05）. The blastocyst diameter obtained with T2 was significantly greater （P 〈 0.05） than with T1 at 7 dpf （173.3 μm ±4.9 μm and 157.2μm ±4.1 μm, respectively）, however, no significant differences were observed at 8 dpf （190.3 μm 5.2 μm and 179.7 μm ± 5.3 μm, respectively）. In conclusion, the synthetic medium （T2） shows a comparable development rate to the control medium and improves the blastocyst diameter and the TCN.