胰岛beta细胞再生研究进展

胰岛beta细胞分泌胰岛素调控体内血糖水平,胰岛beta细胞数量减少会导致糖尿病的发生。胰岛移植是目前治疗糖尿病的有效方法,但是目前仍然面临供体短缺等巨大障碍,因此研究胰岛beta细胞再生对于糖尿病的临床治疗具有深远意义。beta细胞的再生来源主要包括内源性beta细胞增殖、多能干细胞分化和其他非beta细胞的转分化。成体是否存在内源性胰腺干细胞依然是领域内亟待解决的重要科学问题之一。本文总结了与胰岛beta细胞再生相关的研究发现与进展,并讨论了内源性胰岛beta细胞增殖、诱导多能干细胞分化、非胰岛beta细胞重编程等方法在糖尿病治疗中需要注意的问题和潜在应用前景。     

胰岛类器官研究进展

类器官是将具有多向分化潜能的干细胞或组织细胞在特定环境下培养分化成为能够模拟原生器官结构和功能的三维结构。类器官在各种疾病模型研究及药物筛选中发挥至关重要的作用。近年来,通过体外诱导胰腺组织或多能干细胞分化形成具有胰岛细胞功能的胰岛类器官研究成为热点,为胰岛相关疾病模型、药物研究以及糖尿病的治疗提供了新的手段。本文针对胰岛类器官的体外诱导方法及应用前景作一综述。     

TGFB信号通路调节胰岛β细胞增殖的功能研究

该研究主要探讨了转化生长因子β(transforming growth factor β,TGFB)信号通路对胰岛β细胞增殖的影响。以Min6为细胞模型,使用TGFB信号通路激活剂TGFβ1、抑制剂SB-431542分别激活和阻断TGFB信号通路,采用CCK-8细胞活性检测法检测Min6细胞活性,并用流式细胞分选术(fluorescence activated cell sorting,FACS)检测Min6细胞中Ki-67阳性(Ki-67~+)细胞比例。最后用TGFβ1、SB-431542体外处理C57BL/6J小鼠胰岛48 h,免疫荧光检测小鼠胰岛中胰岛素、Ki-67双阳性(Insulin~+Ki-67~+)细胞数量。结果显示,使用TGFβ1处理Min6细胞、小鼠胰岛,Min6细胞活性下降,Ki-67~+ Min6细胞数量减少,小鼠胰岛中Insulin~+Ki-67~+细胞数量减少;使用SB-431542处理Min6细胞、小鼠胰岛,Min6细胞活性上升,Ki-67~+ Min6细胞数量增加,小鼠胰岛中Insulin~+Ki-67~+细胞数量增多。综上所述,抑制TGFB信号通路可促进胰岛β细胞的增殖能力,该研究为胰岛β细胞再生疗法的发展提供了理论支持。     

胰腺干细胞生物学特性研究进展

胰腺干细胞是一类来源于胎儿或成年胰腺组织中的成体干细胞.多数研究认为胰腺干细胞体外培养时,贴壁生长,呈多角形上皮样,具有较高的扩增性;表达胰腺发育过程中的一些决定因子Pdx1、HNF3β、Ngn3、Th及Isll等,还共表达胰腺终末细胞释放的激素glucagon、insulin等,甚至共表达来源于其它胚层细胞的表达物CK19、nestin等;具有多分化潜能,其特点是在自然分化过程中,优先分化为胰腺组织细胞,在特定的条件下,也可转分化为其它组织细胞.胰腺干细胞生物学的不断深入研究,为分离、克隆胰腺干细胞及定向诱导其分化为功能性胰岛移植治疗人类糖尿病奠定了基础.     

干细胞诱导分化为胰岛素分泌细胞的研究现状

胰腺或胰岛细胞移植是目前治疗Ⅰ型糖尿病和部分Ⅱ型糖尿病效果最理想的方法,但因来源组织短缺及需要终生服用免疫抑制剂等问题限制了它的广泛应用.利用胰腺或胰腺外的多能干细胞产生胰岛样细胞有望克服上述问题而用于治疗糖尿病.本文就将干细胞诱导分化为胰岛样细胞中所用的重要的转录因子和可溶性诱导因子及其作用以及胰岛素分泌细胞的来源做一综述.     

利用成体干细胞治疗糖尿病

糖尿病是一类严重的代谢疾病, 正危害着世界上越来越多人口的健康。胰岛移植是一种治疗糖尿病的有效方法,却因供体缺乏和移植后免疫排斥问题制约了其广泛应用。干细胞为具有强增殖能力和多向分化潜能的细胞, 是利用细胞替代疗法治疗重大疾病的细胞来源之一, 其中成体干细胞因不存在致瘤性及伦理道德问题而被人们寄予厚望。成体胰腺干细胞在活体损伤及离体培养条件下均能产生胰岛素分泌细胞, 肝干细胞、骨髓干细胞和肠干细胞等在特定离体培养条件下或经过遗传改造后也均可产生胰岛素分泌细胞, 将这些干细胞来源的胰岛素分泌细胞移植到模型糖尿病小鼠中可以治疗糖尿病。因而, 成体干细胞可以为细胞替代疗法治疗糖尿病提供丰富的胰岛供体来源。     

人乳头状瘤病毒感染与宫颈癌患者临床病理特征和Ki-67、PCNA的关系

目的:研究人乳头状瘤病毒(HPV)感染与宫颈癌患者临床病理特征和Ki-67、细胞增殖抗原(PCNA)的相关性,从而为临床宫颈癌的诊治提供参考依据。方法:选取2016年3月～2018年6月于我院接受手术治疗的宫颈病变患者130例为研究对象。其中宫颈癌患者30例记为宫颈癌组,宫颈上皮内瘤变患者68例记为宫颈上皮内瘤变组,慢性宫颈炎患者32例记为对照组。采用免疫组织化学法检测各组宫颈组织中HPV感染、Ki-67以及PCNA阳性表达情况,并分析HPV与宫颈癌患者临床病理特征的关系及其与Ki-67、PCNA的相关性。结果:宫颈癌组、宫颈上皮内瘤变组患者HPV、Ki-67以及PCNA阳性率均高于对照组,宫颈癌组高于宫颈上皮内瘤变组(均P0.05)。临床分期Ⅲ～Ⅳ期以及淋巴结转移宫颈癌患者HPV感染率均明显高于临床分期Ⅰ～Ⅱ期与无淋巴结转移患者(均P0.05)。经Spearman相关性分析可得:宫颈癌患者HPV感染与Ki-67、PCNA表达均呈正相关关系(均P0.05)。结论:宫颈癌患者存在明显的HPV感染,且HPV感染与宫颈癌患者临床分期、淋巴结转移、Ki-67、PCNA表达存在一定相关性,临床可通过对HPV、Ki-67、PCNA进行联合检测,从而有助于宫颈癌的早期诊断。     

miR-148a过表达抑制胰腺祖细胞增殖及其机制研究

胰腺祖细胞在糖尿病治疗中有着巨大的潜能,其增殖分化受到microRNAs等多种机制调控。该文探究了miR-148a水平升高对胰腺祖细胞增殖的影响及可能的机制。结果显示,miR-148a在胰腺祖细胞分化过程中升高,表明其可能参与胰腺的发育进程。光镜观察和免疫染色检测表明,过表达miR-148a可以显著抑制胰腺祖细胞的增殖;流式检测结果显示,miR-148a使细胞停滞在S期;Western blot结果显示,miR-148a不是通过诱导凋亡来抑制细胞增殖,而很可能是通过抑制AKT信号通路来抑制胰腺祖细胞的增殖;q RT-PCR结果表明,过表达miR-148a可以上调PTEN的mRNA表达水平。以上研究表明,过表达miR-148a可能通过上调PTEN来抑制AKT通路,从而实现对胰腺祖细胞增殖的抑制。此研究为今后利用microRNAs抑制剂治疗胰腺疾病提供理论基础,也为利用microRNAs治疗胰腺疾病提供线索。     

脐带间充质干细胞在糖尿病治疗中的研究进展

糖尿病是一种以高血糖为特征的代谢性疾病,胰岛β细胞功能衰竭和胰岛素抵抗是糖尿病的主要病因。然而,目前的治疗方法,如口服抗糖尿病药物及胰岛素注射,尚不能逆转胰岛β细胞功能衰竭,而胰腺移植又受到供体来源的限制。随着干细胞与再生医学的发展,脐带间充质干细胞(umbilical cord mesenchymal stem cells,UC-MSCs)由于来源丰富、免疫原性低、无伦理问题等,成为治疗糖尿病的理想细胞类型。现就UC-MSCs在糖尿病治疗中的研究进展进行综述,为UC-MSCs移植治疗糖尿病的临床应用研究提供参考。     

干细胞定向分化胰岛β细胞新进展

在治疗糖尿病领域里,干细胞定向分化成胰岛β细胞是目前新颖又有前景的一种糖尿病治疗策略,不仅克服了注射胰岛素所带来的并发症,还避免了胰岛移植供体来源的短缺不足。目前供体细胞的材料主要有从胰腺中分离出的胰腺干细胞/胰腺祖细胞、胰腺导管细胞、胰腺泡细胞、肝实质细胞、小肠细胞、神经干细胞、ES细胞以及近来研究热点之一的iPS细胞。以上这些细胞都被证明或多或少具备分化成胰岛素分泌的细胞形态的潜力。在体外分化技术方面,国际上有D’Amour法、Lumelsky法等。现对干细胞定向分化胰岛β细胞的一些研究概况作简单评述。     

Measurement of cell proliferation in gastric carcinoma: comparative analysis of Ki-67 and proliferative cell nuclear antigen (PCNA)

Summary Immunostaining to identify nuclear antigens expressed throughout the cell cycle provides a convenient way of assessing proliferating kinetics in tumours. We studied proliferation activity of gastric carcinomas by Ki-67 and PCNA immunostaining and the two methods were compared. The mode of tissue preparation differed, fresh frozen for Ki-67 and formalin-fixed paraffin-embedded for PCNA. Immunostaining with avidin-biotin was used in both. The labelling index (LI) and a semi-quantitative grading of cell proliferation were assessed in both markers. Significant correlation was shown between LI and grading with either Ki-67 and PCNA. However, no correlation was found between PCNA and Ki-67. This lack of relationship between the two markers may be attributed to a number of factors. 1. The most likely is the marked inter- and intra-tumour heterogeneity of gastric carcinomas reflected in high standard deviation values. 2. Preparation of tissue and small size sampling with Ki-67. 3. Long life of PCNA leading to detection of cells that have recently left the cell cycle. 4. One may be observing deregulated expression of DNA as seen in certain tumours. PCNA offers the advantage of being applicable to archival material.     

Biomarkers and molecular imaging in gastroenteropancreatic neuroendocrine tumors

Neuroendocrine gastrointestinal and pancreatic tumors (GEP-NETs) are a heterogenous group of cancers with various clinical expressions. All tumors produce and secret various amines and peptides, which can be used as tissue and circulating markers. Chromogranin A (CgA) is a general tumor marker stored in secretory granules within the tumor cell and released upon stimulation. CgA is the best general tumor marker at the moment, expressed in 80-90% in all patients with GEP-NETs. CgA and NSE are used as tissue markers for the delineation of the neuroendocrine features of the tumors, but recently also the proliferation marker Ki-67 has been included in the standard procedure for evaluation of the proliferation. GEP-NETs are classified into well differentiated neuroendocrine tumors (Ki-67<2%), well-differentiated neuroendocrine carcinoma (Ki-67 2-20%), poorly differentiated neuroendocrine carcinoma (Ki-67>20%). The molecular imaging of NETs is based on the ability of these tumor cells to express somatostatin receptors as well as the APUD features. Octreoscan has been applied for imaging and staging of the disease for more than 2 decades and will nowadays be replaced by 68Ga-DOTA-Octreotate, with higher specificity and sensitivity. 18Fluoro-DOPA and 11C-5HTP are specific tracers for NETs with high specificity and selectivity. A new potential biomarker is auto-antibodies to paraneoplastic antigen MA2, which might indicate early recurrence of carcinoids after surgery with a curative intent. Circulating tumor cells (CTC) have been applied in GEP-NETs quite recently. There is still an unmet need for new markers.     

PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.     

Histone H3 mRNA in situ hybridization for identifying proliferating cells in human pancreas, with special reference to the ductal system

In general, the incidence of proliferating cells parallels that of carcinogenesis. We have investigated proliferating activity and phenotype expression in epithelial cells in normal tissue, mucinous metaplasia and ductal adenocarcinoma of the pancreas. Twenty-eight resected pancreases (15 cases of pancreatic ductal adenocarcinoma and 13 cases of other diseases) were examined. Formalin-fixed, paraffin-embedded tissue sections were examined for proliferating cell activity using histone H3 mRNA in situ hybridization and immunostaining for Ki-67. In the normal pancreas, the labelling indices for proliferating cells were low and no generating zone was found. The following progressive increase was found in the labelling indices: normal ductal epithelium < mucinous metaplasia without papillary hyperplasia < mucinous metaplasia with papillary hyperplasia < ductal carcinoma. In the pancreatic ductal adenocarcinomas, the S-phase fraction, as defined by the ratio H3-mRNA-labelling index/Ki-67-labelling index, increased as the degree of differentiation decreased. Mucinous metaplasia with papillary hyperplasia showed organoid differentiation toward pyloric mucosa. If used in combination with other proliferative markers on paraffin-embedded tissue sections, histone H3 mRNA in situ hybridization could open broader perspectives on the biology of cell proliferation in the pancreatic ductal system.     

Expression of p120, Ki-67 and PCNA as proliferation biomarkers in imprint smears of prostate carcinoma and their prognostic value

The cell proliferation markers p120, Ki-67 and proliferating cell nuclear antigen (PCNA) recognize nuclear antigens. The expression of these proteins by immunostaining methods was reported to be of value in determining the prognosis of patients with malignant diseases. In this study, we evaluated the prognostic significance of the expression of nuclear antigens p120, PCNA and Ki-67 in prostate cancer and compared the results with other prognostic factors. Imprint smear samples obtained from 70 patients immediately after radical prostatectomy for prostatic carcinoma were immunostained with monoclonal antibodies against p120, Ki-67 and PCNA. The immunostaining results were correlated with Gleason score, tumour differentiation, stage and prostatic specific antigen (PSA) levels. Our findings demonstrate that p120, Ki-67 and PCNA expression in prostatic carcinoma smears, correlated significantly with the degree of Gleason score (P < 0.001). When combining p120, Ki-67 and PCNA positivity with tumour differentiation there was a significant association among these parameters (P < 0.001). Overexpression of p120, Ki-67 and PCNA, was also associated with increased PSA serum levels (>4 ng/ml) (P < 0.001). The distribution of p120, Ki-67 and PCNA expression in prostate carcinomas was not statistically significant for Ki-67 (P = 0.69) and p120 (P = 0.22) but was significant for PCNA (P < 0.001) as far as the histological stage (T2a, T2b, T2c, T3a). P120, Ki-67 and PCNA expression had significant prognostic value for disease-free survival. Our results conclude that nuclear antigens p120, Ki-67 and PCNA appear to be additional markers in the field of prognosis of prostatic carcinoma.     

The Ki-67 protein: from the known and the unknown

The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti-Ki-67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease. The best-studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni- and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki-67 equivalent protein from rodents now extends the use of the Ki-67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki-67 protein. Our current knowledge of the Ki-67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell-division cycle is summarized and discussed. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle.     

Assessment of proliferative activity of thyroid Hürthle cell tumors using PCNA, Ki-67 and AgNOR methods

We have undertaken an attempt to compare the application efficacy of the proliferative activity markers in differential diagnosis of thyroid Hürthle cell tumors (HCT) using the PCNA and Ki-67 labeling and AgNOR visualisation techniques. The present work is a retrospective analysis of 78 Hürthle cell tumors: 20 Hürthle cell carcinomas (HCC), 32 Hürthle cell adenomas (HCA) and 26 hyperplastic nodules with Hurthle cell metaplasia (HCM). Five microm sections were stained according to AgNOR technique and labeled with antibodies against PCNA and Ki-67. AgNOR dot count in the nucleus and proliferative index (PI - percentage of cells expressing PCNA and Ki-67) in randomly chosen nuclei (100 in case of AgNOR and over 1000 in case of PI) were evaluated in each slide. The mean values of AgNOR dot count, PI-PCNA and PI-Ki-67 in HCC, HCA and HCM were respectively: 5.1, 61.3 and 54.9; 3.4, 42.4 and 38.6 and 2.5, 39.3 and 34.3. Statistically significant difference was found in all the proliferative activity markers between malignant and benign tumors: HCC:HCA (p<0.01) and HCC:HCM (p<0.001). There was no statistically significant difference between HCA and HCM.     

Proliferation index and karyometric features of pancreatic intraductal proliferative lesions.

The increasing frequency and poor prognosis in pancreatic cancer prompt us to search for morphological lesions being a substrate for its development. Studies of autopsy and surgically resected material as well as recent molecular studies have proved that one of the possible pathways of pancreatic neoplasia is the intraepithelial proliferation--dysplasia--cancer sequence. In the present paper we studied the proliferative activity (Ki-67 index) in pancreatic intraepithelial proliferative lesions and its correlation with geometric features of cell nuclei as signs of increasing dysplasia. The studies were carried out in a group of 35 patients operated on for pancreatic cancer, chronic pancreatitis and other conditions not associated with the pancreas. We used immunohistochemical methods and basic morphometric parameters. The results of our studies indicate that the cell proliferative activity depends both on the type of epithelial proliferation and underlying pancreatic disease. The values of Ki-67 index are significantly different in low-grade proliferation (flat and papillary hyperplasia) and high-grade proliferation (atypical papillary hyperplasia and carcinoma in situ). A set of karyometric features correlates with Ki-67 index but there is no single feature which would have a diagnostic value.     

PCNA Ki-67 dissociation in childhood acute lymphoblastic leukaemia. An Immunofluorescent Laser Confocal Scanning Microscopical study.

Cell proliferation rates of diagnostic marrow aspirate cells of 21 children with Acute Lymphoblastic Leukaemia and 16 controls were compared using immunocytochemical labelling of PCNA and Ki-67 antigen as assessed by Confocal Laser Scanning Microscopy. The results showed an unexpected, highly significant degree of dissociation between PCNA and Ki-67 expression in ALL blasts. The PCNA labelling indices of ALL patients were significantly increased (mean 44, range 24-77) compared with normal reactive marrow cells (mean 13.8, range 4-26) (p<0.000001, Mann Whitney U two tailed test), suggesting an abnormal commitment to proliferation. Ki-67 expression was raised to a lesser extent in ALL cells (mean 14.8, range 1.2-35) when compared to non-malignant proliferations (mean 6.6, range 1.7-25) (p < 0.02). PCNA/Ki-67 LI ratios in ALL (mean 7, range 1.1-35) were higher than in controls (mean 2.7, range 1.04-6.5, p<0.09). As cell proliferation rates actually achieved in the bone marrow do not differ as strongly as suggested by the extreme difference in PCNA labelling, a pathological dissociation of PCNA / Ki-67 expression exists, suggesting immortalisation.