组织化胰腺的体外构建及其在细胞移植中的作用

目的探讨组织工程化胰腺的体外构建及其在细胞移植中的作用。方法取SD大鼠8只,采用旋转壁式生物反应器,建立微重力环境下共培养SD大鼠胰岛和睾丸支持细胞,建立糖尿病SD大鼠模型,进行胰岛移植。移植后,检测各组大鼠血糖水平,摘取30 d以上保持血糖正常水平的大鼠肾脏组织,清洗后固定,包埋、切片,进行苏木素-伊红(HE)染色,显微下观察,并进行Fas-L、胰岛素免疫组化学法染色,对大鼠移植物进行鉴定并检测葡萄糖糖耐量。结果分离纯化的睾丸支持细胞贴壁后于培养箱中继续培养24 h,细胞基本已全部贴壁生长,细胞立体感较强,显微镜下可见极少量球形精细胞贴附于睾丸细胞上,经台盼蓝染色后活细胞透亮,总数占95%以上。HE染色可见细胞形态呈宽大柱状或纤维样,排列紧密,多个一起存在时融合成片,界限不清,呈单层膜状,一般有3~4个突起,突起相互连接,胞质铺展较大,可见多量大小不一的空泡状结构。培养24 h后,睾丸支持细胞-胰岛球形细胞复合物逐步形成,复合物呈球形,被支持细胞包裹;Fas-L免疫组织切片染色可见支持细胞呈阳性,与胰岛素阳性的β细胞检测结果与透射电镜观察结果一致,共培养的胰岛生长状态相对较好。从血糖水平结果总结,微重力环境下共培养睾丸支持细胞-胰岛细胞复合物能够提高胰岛活性,降低其免疫原性;与接受新鲜胰岛移植的SD大鼠相比,复合物移植使模型大鼠保持血糖正常时间明显更久。结论移植睾丸支持细胞-胰岛细胞复合物的模型大鼠体内胰岛对葡萄糖保持明显的敏感性,复合物移植组大鼠平均血糖基本在2 h内可以恢复正常,与正常检测结果接近。

用猪、家兔、大鼠、小鼠和豚鼠胰腺做抗原免疫组织化学检测ICA的比较研究

免疫组织化学检测Ⅰ型糖尿病（ＩＤＤＭ）病人血清中的胰岛细胞抗体（ＩＣＡ）需用“Ｏ”型血人胰腺做抗原。因为人胰腺来源困难，所以，本研究选用猪、家兔、大鼠、小鼠和豚鼠以及“Ｏ”型血正常人胰腺作抗原，对１１例ＩＤＤＭ患者和１０例正常人血清，进行ＩＣＡ免疫组织化学检测，以期筛选寻找ＩＣＡ免疫组织化学检测的最佳抗原替代品。选用已确诊为ＩＤＤＭ并用“Ｏ”型血正常人胰腺作抗原ＩＣＡ检测为阳性患者的血清１１份，正常人血清１０份，同步用ＰＯＰＡ免疫组织化学技术进行检测。结果发现：在１１份ＩＤＤＭ患者血清中，用猪胰腺与用“Ｏ”型血正常人胰腺作抗原，１１例均为阳性，而用家兔和大鼠胰腺作抗原各有１０例为阳性。但用小鼠和豚鼠胰腺作抗原，则分别只有２例和１例呈阳性。在１０例正常人血清中，人和各种动物胰腺均呈阴性反应。本研究结果表明猪胰腺是ＩＣＡ免疫组织化学最佳替代品，其次是家兔和大鼠的胰腺。

Endoscopic ultrasound-guided fine needle aspiration in the differentiation of type 1 and type 2 autoimmune pancreatitis

AIM:To investigate the usefulness of endoscopic ultrasound-guided fine needle aspiration(EUS-FNA) in the differentiation of autoimmune pancreatitis(AIP).METHODS:We retrospectively reviewed 47 of 56 AIP patients who underwent EUS-FNA and met the Asian diagnostic criteria.On 47 EUS-FNA specimens,we evaluated the presence of adequate material and characteristic features of lymphoplasmacytic sclerosing pancreatitis(LPSP) and idiopathic duct-centric pancreatitis(IDCP) mentioned in the International Consensus Diagnostic Criteria and examined if these findings make a contribution to the differential diagnosis of type 1 and type 2 AIP.A disposable 22-gauge needle was used for EUS-FNA.RESULTS:Adequate specimens including pancreatic tissue for differentiating AIP from cancer were obtained from 43 of 47 patients who underwent EUSFNA.EUS-FNA was performed from the pancreatic head in 21 cases,which is known to be technically difficult when performed by core biopsy;there was no significant difference in the results compared with pancreatic body-tail.Nine of 47 patients met level 1 findings of LPSP and 5 patients met level 2 findings of LPSP.No one met level 1 findings of IDCP,but 3 patients met level 2 findings of IDCP.Of 10 seronegative cases,2 cases were diagnosed with "definitive type 1 AIP",and 3 cases were diagnosed with "probable type 2 AIP" when considering both the level 2 histological findings and response to steroids.CONCLUSION:EUS-FNA is useful in the differentiation of type 1 and type 2 AIP,particularly in seronegative cases.

Expression of cell cycle regulator p57^(kip2), cyclinE protein and proliferating cell nuclear antigen in human pancreatic cancer: An immunohistochemical study

AIM： To investigate the effects of p57^kip2, cyclinE protein and proliferating cell nuclear antigen （PCNA） on occurrence and progression of human pancreatic cancer. METHODS： The expression of p57^kip2, cyclinE protein and PCNA in tumor tissues and adjacent tissues from 32 patients with pancreatic cancer was detected by SP immunohistochemical technique. RESULTS： The positive expression rate of p57^kip2 protein in tumor tissues was 46.9%, which was lower than that in adjacent pancreatic tissues （χ^2 = 5.317, P〈0.05）. p57^kip2 protein positive expression remarkably correlated with tumor cell differentiation （P〈0.05）, but not with lymph node metastasis （P〉0.05）. The positive expression rate of cyclinE protein in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues （χ^2 = 4.063, P〈0.05）. CyclinE protein positive expression significantly correlated with tumor cell differentiation and lymph node metastasis （P〈0.05）. The positive expression rate of PCNA in the tumor tissues was 71.9%, which was higher than that in adjacent pancreatic tissues （χ^2 = 5.189, P〈0.05）. PCNA positive expression remarkably correlated with tumor cell differentiation and lymph node metastasis （P〈0.05）. CONCLUSION： The decreased expression of p57^kip2 and/or overexpression of cyclinE protein and PCNA may contribute to the occurrence and progression of pancreatic cancer. p57^kip2, cyclinE protein, and PCNA play an important role in occurrence and progression of pancreatic cancer.

Increased expression of neuropeptide Y and its mRNA in STZ-diabetic rats

OBJECTIVE: To study the relationship between neuropeptide Y (NPY) and diabetes by examining the content and distribution of NPY and its mRNA expression in the hypothalamus and pancreas of STZ-diabetic rats. METHODS: Thirty Wistar rats were randomly divided into 3 groups (diabetic group, diabetic insulin treatment group, and control group). After feeding for 24 weeks, the rats were sacrificed. The expression of NPY in the hypothalamus and pancreas was detected with immunohistochemistry and in situ hybridization. RESULTS: (1) The hypothalamic content of NPY and its mRNA were significantly increased in STZ-diabetic rats in comparison with normal controls. Increased expression of NPY mRNA was found only in the arcuate nucleus and not in the paraventricular nucleus in diabetic rats, suggesting that NPY was produced in the arcuate nucleus. (2) The hypothalamic content of NPY and its mRNA in STZ-diabetic rats were visibly reduced after insulin treatment compared with that in untreated diabetic rats. This supports the hypothesis that insulin deficiency in the brain may be responsible for increased hypothalamic NPY gene expression in diabetic rats. (3) The increase of hypothalamic NPY in STZ diabetic rats associated with hyperphagia and polydipsia could be reversed by insulin replacement, suggesting that increased hypothalamic NPY contributes to the pathophysiological progress of the diabetic state. (4) The present study demonstrated for the first time that the content of NPY and its mRNA in the pancreas was increased in STZ-diabetic rats, and that the distribution of NPY-positive cell in islets was changed from the periphery to the whole islet. The content and distribution of NPY and its mRNA in islets were not changed by insulin treatment. CONCLUSION: Increased NPY in the hypothalamus results in hypophagia and polydipsia, while the implication of increased NPY in the pancreas of diabetic rats is not clear.

Cell therapy in diabetes: current progress and future prospects

Diabetes mellitus, characterized by the impaired metabolism of insulin secretion in β cells, is becoming one of the most prevalent diseases around the world. Recently, cell replacement based on differentiation of various pluripotent stem cells, including embryonic stern cells, induced pluripo- tent stem cells and multipotent stem cells, such as bone mar- row mesenchymal stem cells, adipose-derived stem cells and gnotobiotic porcine skin-derived stem cells, is becoming a promising therapeutic strategy. Cells derived from pancreatic tissues or other tissues that are relevant to β cell differentiation have also been used as cell source. However, in spite of hopeful experimental results, cell therapy in diabetes still confronts certain obstacles, such as purity of cells, functional differentiation of stem cells and possible tumorigenesis, which, in turn, lead to the seeking of new-generation tools, such as xenogenetic materials. In this review, we will sum- marize the current knowledge and future prospects of cell therapy in diabetes mellitus.

Anti-tumor effect of the extract from Qingyihuaji formula on pancreatic cancer by down-regulating Notch-4 and Jagged-1

OBJECTIVE： To investigate, in terms of Notch signaling pathway, the effect on pancreatic cancer of the extract of an anti-tumor prescription -- Qingyihuaji formula （QYHJ） -- from Traditional Chinese Medicine （TCM）.METHODS： Nude mice were implanted subcutaneously with human pancreatic cancer cell line SW1990 and then randomly divided into four groups： Control, QYHJ extract, Gemcitabine, and Combination of QYHJ extract and gemcitabine. Treatments were given for 21 days and tumor growth was evaluated simultaneously. Then, expression of Notch receptors （Notch-I, Notch-2, Notch-3, and Notch-4） and their Jagged ligands （Jagged-1 and Jagged-2） in dissected tumor tissue were detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot. Finally, immunohistochemistry was performed to detect CD133, a marker of pancreatic cancer stem cells （CSCs）, to evaluate the impact of QYHJ extract on pancreatic CSCs.RESULTS： QYHJ extract treatment effectively inhib- ited the tumor growth in nude mice. The expression of both Notch-4 and Jagged-1 were decreased significantly in QYHJ treatment groups （P 〈 0.05）, while gemcitabine alone had no significant effect in down-regulating Jagged-1 （P 〉 0.05）. No significant difference was observed in the ex- pression of Notch-1, Notch-2, Notch-3, and Jagged-2 between three treatment groups and control group （P 〉 0.05）. Moreover, immunohistochemical analysis showed that the number of CD133 positive cells was significantly reduced by QYHJ treatment （P 〈 0.05）, and the combined treatment was more effective than gemcitabine alone （P 〈 0.05）.CONCLUSION： The role of the extract in pancreatic cancer treatment was associated with down-regulation of Notch-4 and Jagged-1 in Notch signaling pathway. The extract could enhance the antitumor activity of gemcitabine and was more effective than gemcitabine in regulating Notch signaling pathway to some extent.