鼻咽癌及癌旁上皮细胞的AgNORs定量分析及其生物学意义

目的通过鼻咽癌及癌旁上皮的细胞核仁组成区蛋白(NORs)图像定量分析,评价其反映细胞群体增生能力及分化程度等生物学意义。方法对70例鼻咽癌石蜡切片进行银染(AgNORs),应用CAS200图像分析仪分别测定29例癌旁正常腺上皮、19例增生／异型增生柱状上皮、10例增生／异型增生鳞状上皮、54例分化性非角化性癌和16例未分化癌的细胞群体AgNORs参数值并作比较分析;结果每核AgNOR计数、每核AgNOR面积和平均AgNOR面积／粒从正常腺上皮至增生／异型增生柱状上皮至分化性非角化性癌或未分化癌呈现显增加,平均核面积、每核AgNOR面积和平均AgNOR面积／粒从正常腺上皮至增生／异型增生鳞状上皮至未分化癌呈现显增加,差异均有显性,但增生／异型增生鳞状上皮与分化性非角化性癌的AgNOR数值差异没有显差异;与分化性非角化性癌比较,未分化癌的平均核面积、每核AgNOR面积和平均AgNOR面积／粒显增加。结论．AgNORs形态定量分析反映了鼻咽癌及癌旁不同类型鼻咽上皮的细胞增生活性和组织细胞分化程度,但不能反映细胞是否恶性转化;不同组织类型鼻咽癌细胞存在增殖能力的差异,其增生差异程度可能是影响病人对放射线治疗敏感性及其预后的重要因素之一。     

长期培养人脐带间充质干细胞分化能力改变的研究

目的:分析体外传代培养至23代,人脐带间充质干细胞(human umbilical cord-MSCs, hUC-MSCs)多向分化能力的改变,以探索hUC-MSCs体外诱导分化的最佳时间窗。方法:采用胶原酶消化法提取hUC-MSCs,并用胰酶消化传代;收集不同代细胞,用流式细胞术检测细胞表面抗原及细胞周期;MTT法检测不同代细胞的增殖活性;取不同代细胞进行成骨、成软骨及成脂肪诱导鉴定;并利用实时定量PCR分别检测OCT-4、SOX-2、Nanog的mRNA表达水平。结果:用胶原酶消化法可获得形态均一,可稳定传代23代以上的hUC-MSCs;体外传代培养至23代,细胞表面标志物表达率无明显改变;细胞生长曲线形态相似;细胞周期亦无明显差异,(73.04±1.15)%的细胞处于G0/G1期;细胞均可被诱导成骨细胞、软骨细胞和脂肪细胞;不同代细胞OCT-4、SOX-2、Nanog 的mRNA表达无显著差异。结论:体外培养至23代,随着传代次数的增加,hUC-MSCs的多向分化能力并未受影响。     

胎盘间充质干细胞的体外培养及其生物学特性研究

目的：探讨胎盘间充质干细胞（PMSCs）的体外分离和培养方法,建立稳定的PMSCs体外培养扩增体系。方法：将胎盘组织经胶原酶消化、密度梯度离心、贴壁筛选法分离,获得并培养人PMSCs,观察细胞形态及其超微结构;应用流式细胞术测定细胞周期及CD14、CD29、CD34、CD44、CD45的表达,研究其增殖和生长特性。结果：在体外培养条件下,人PMSCs贴壁生长,为成纤维细胞样,与骨髓间充质干细胞相似;CD14/CD34/CD45阴性,CD29/CD44阳性,核浆比大,细胞周期检测G0/G1期约占95%,具有原始细胞的特征。结论：体外获得的PMSCs形态单一、生长稳定、增殖能力较强,具有与骨髓间充质干细胞相似的细胞形态、表面标志。由于其来源方便、丰富,无伦理学限制,因此可进一步用于细胞治疗的研究。     

嵌合抗原受体（CAR）-T细胞的单细胞转录组测序研究

CAR-T细胞疗法是一种新兴的肿瘤免疫疗法,目前已经在白血病、骨髓瘤等血液系统肿瘤的治疗中取得显著疗效。由于CAR-T细胞是在体外诱导产生的细胞群体,具有很高的异质性。这种异质性的存在,是影响CAR-T细胞治疗效果的重要因素。本研究通过单细胞转录组测序技术,对基于NKG2D分子识别和第3代CAR-T细胞技术构建的NKG2D/CAR-T细胞进行了转录组学特征的分析。结果表明,通过对比特征基因进行降维聚类,将NKG2D/CAR-T细胞分为12个细胞簇,其特征基因显示NKG2D/CAR-T细胞以幼稚样和中央记忆样细胞亚群为主。不同细胞簇表达基因存在差异,对差异基因进行富集分析发现,这些差异基因富集在病毒侵入细胞、插入宿主细胞DNA并复制、T细胞代谢、增殖、活化及分化等信号通路。     

中老年人骨髓间质干细胞的生物学特性研究

目的:研究中老年人骨髓间质干细胞(MSCs)的生物学特性。方法:用Ficoll-Paque分离不同年龄段供者MSCs体外培养,进行诱导分化,用流式细胞术检测表面标志,核型分析观察细胞染色体的稳定性,裸鼠实验、软琼脂实验观察致瘤性。结果:中老年人每毫升骨髓分离的单个核细胞量和MSCs增殖速度与青年人无显著差异;中老年人不同代次的MSCs诱导后具有向成骨细胞和成脂细胞分化的能力;分离培养的MSCs表达CD44,不表达CD34;传至第10代核型未见异常,未见致瘤性。结论:中老年人MSCs有较强的体外增殖能力和多向分化潜能,遗传背景稳定。     

稻胚发育过程中细胞核内DNA增殖的特点

应用显微分光光度技术测定的稻胚发育过程中核DNA含量的结果表明,球形胚中细胞核DNA含量较高,多倍性明显,C值较集中在6－12C,个别的最高达238或439C,它们可能为胚柄细胞;DNA含量差异明显可能与它们的分化潜能有关。分化初期、中期及成熟胚细胞核DNA合量一般不及球形胚高,不同倍性的频率分布亦较球形胚分散,但多倍性仍明显;胚分化完全后胚细胞核DNA含量较多变动在20－56C范围内,但盾状体细胞核DNA含量明显高于胚芽、胚根原基。说明胚器官原基间核DNA的增殖或复制特点不同。核DNA含量高和不同器官原基间差异复制的不同可能是胚分化的前兆和基础。     

脂肪来源细胞体外增殖规律及定向诱导分化研究

脂肪组织由整形外科吸脂术获得(19例,31．5±5．8岁)。酶消化法分离抽吸物中细胞,体外扩增至第10代．测定细胞生长曲线、累计倍增数目,明确其体外生长规律和增殖能力;通过对表面抗原CD29、CD105、CD106、CD166、CD49d、CD34、CD31、3G5等的检测分析脂肪来源细胞的群体组成：分别向软骨、骨、脂肪定向诱导,进一步明确该细胞群体定向分化能力。实验表明,每300ml脂肪抽吸物平均可获得5×10~7个有核细胞,体外扩增10代,平均每代倍增数目为1．59±0．224．累计倍增数目为15．53。流式细胞学及免疫细胞化学检测显示,干细胞相关抗原CD29、CD105、CD106、CD166等表达率均＞60%,但与造血系相关的CD34、CD31表达率也分别达到7．3%、29．2%。ADC向软骨诱导可检测到Ⅱ型胶原表达;向成骨诱导可见矿化结节形成,并可检测到AKP、Osteonectin基因表达;向脂肪诱导可检测到PPARr2、GLU-4、Leptin基因表达,细胞内有脂滴形成。脂肪来源的细胞获得量大,体外增殖能力强,并含有具有多向分化潜能细胞,有可能作为组织构建的种子细胞。     

一株新的人肾癌原代细胞系的建立及其生物学特性鉴定

目的:取肾癌病人标本建立一株新的人肾癌细胞系,初步对该细胞系进行鉴定,为进一步的肾癌基础研究提供实验模型.方法:2008-2009年间共采集20例肾癌新鲜手术标本,于手术后1 h内将每例标本切取四块0.5cm× 0.5 cm× 0.5 cm组织块,分别包埋于2只裸鼠的右后肢皮下及背部皮下,连续传代3次,取移植瘤体外培养,记录细胞株的生长曲线,测定细胞集落形成率,对细胞进行DNA含量测定,进行染色体分析及病理学检查.结果:其中1只裸鼠皮下移植瘤生长,继续传代,肿瘤生长速度明显加快.取移植瘤标本体外培养得到肾癌细胞系XJG-9201.形态结构,分化程度与原发瘤一致,染色体众数为65.细胞群体倍增时间为38.2h,细胞周期分析G1期62.7%,G2期11.2%,S1期25.3%,集落形成率为70%.结论:肾癌细胞系XJG-9201与原发癌保持相同的生物学特性,体外连续传代1年以上传115代.细胞形态不变,生长周期恒定,已成为一个稳定的细胞系.     

包囊游仆虫包囊形成和解脱过程中大、小核的研究

包囊游仆虫形成包囊时大核除经历形态大小的变化外,大核DNA含量也低于正常大核水平;细胞脱包囊前,在大核一端或两端发生染色质粗浓集,是大核DNA复制的结果;染色质粒在整个大核内浓集时,大核DNA含量已达到正常大核水平,此时DNA复制结束,细胞脱包囊。小核在游仆虫形成包囊和脱包囊过程中,其形态大小、DNA含量等无明显变化。     

牛肝干细胞的分离培养与鉴定

该实验旨在分离牛肝干细胞,培养观察其生长特性,为比较医学研究及开发牛肝脏体外研究工具提供实验基础。实验采用改良的离体两步胶原酶灌流法,结合密度梯度离心分离牛肝干细胞,观察细胞形态及生长特性,并利用免疫荧光染色和PCR对牛肝干细胞相关标志物进行鉴定。实验分离的细胞接种48 h开始贴壁分裂,随后呈集落样生长,表现出干细胞的特性,低密度下培养504 h后细胞呈肝细胞样分化。正常接种密度的细胞可传至P3代,HE染色可见细胞均为单核,核浆比大,呈幼稚低分化状态;免疫荧光染色和PCR结果均显示,该细胞表达甲胎蛋白AFP、上皮细胞黏附蛋白(EpCAM)、造血干/祖细胞表面标志CD34、干细胞因子受体蛋白(c-kit)、细胞角蛋白CK18、CK19。结果表明,该研究分离了一种具有明显干细胞特征,并表达成熟肝细胞和胆管上皮细胞表面标志物的牛肝干细胞。     

Growth kinetics as a function of ploidy in diploid, tetraploid, and octaploid smooth muscle cells derived from the normal rat aorta

The smooth muscle cell population in major arteries of humans and experimental animals is heterogeneous with regard to cellular DNA content. A proportion of cells has polyploid DNA content and this proportion increases with normal aging and with hypertension. We have isolated pure populations of rat aortic smooth muscle cells containing 2C, 4C, and 8C DNA content by cloning of cultures of cells previously subjected to flow cytometric cell sorting. Karyologic analysis of these clonal populations revealed them to be pure diploid, tetraploid, and octaploid populations, respectively, containing 2N (= 42), 4N, and 8N chromosomes. Cell attachment area and nuclear size appeared to increase with the level of ploidy. Studies of the proliferative characteristics of the cells revealed that the growth rate and ultimate cell densities achieved decreased as the ploidy level increased. The intrinsic cellular radiosensitivity of these clones did not vary with ploidy. Increased smooth muscle cell ploidy is, therefore, associated with a decreased rate of proliferation. The emergence of smooth muscle cells with polyploid DNA content under normal and pathologic conditions is probably due to mitotic polyploidization without net cell proliferation and may be related to the need for expression of differentiated functions.     

含EBV-LMP2基因重组腺病毒修饰的树突状细胞疫苗体内外特异性抗瘤作用的研究

鼻咽癌(NPC)在东南亚地区,尤其在我国南方地区发病率高达40／10万～60／10万。Epstein—Barrvirus(EBV)在鼻咽癌的病因学中有着十分重要的作用,在肿瘤细胞中该病毒的存在为以CTL为基础的免疫治疗提供了一个潜在靶位。过继性EBV特异性CTL,成功地治疗了骨髓移植受者体内出现的来自捐献者的免疫增生性B细胞淋巴瘤的研究,显示了这种方法的可行性。LMP2已经被证实可以诱发较强的特异性细胞免疫反应,而且也确定了很多CTL表位。目前许多关于EBV相关肿瘤治疗的研究都是针对LMP2的。树突状细胞(DC)是人体内最强大的抗原呈递细胞(APC),在肿瘤的免疫治疗中有着重要的作用。     

5-杂氮-2′-脱氧胞苷诱导鼻咽癌细胞株Syk基因启动子去甲基化的研究

利用5-杂氮-2′-脱氧胞苷（5-aza-2′-deoxycytidine,5-aza-CdR）处理体外培养的鼻咽癌细胞株CNE-1、CNE-2及永生化非癌性人鼻咽上皮细胞株NP-69,采用BS-PCR、Q-RT-PCR及Westernblot方法分别检测经5μmol/L的5-aza-CdR处理前后,各细胞株中Syk基因启动子甲基化状况及SykmRNA和蛋白质表达情况。探讨去甲基化药物5-杂氮-2′-脱氧胞苷（5-aza-CdR）对鼻咽癌细胞株中脾酪氨酸激酶（spleen tyrosine kinase,Syk）启动子甲基化水平及其表达的影响。结果显示,Syk基因启动子甲基化水平与鼻咽癌细胞分化程度呈负相关,两种鼻咽癌细胞株的Syk mRNA和蛋白质表达水平显著低于NP-69细胞（P〈0.01）;经5-aza-CdR处理后两种鼻咽癌细胞株的Syk基因启动子甲基化水平降低,Syk mRNA及蛋白质表达升高（P〈0.05）;高分化鼻咽癌细胞株对药物敏感性高于低分化鼻咽癌细胞株（P〈0.01）。由此可见,两种鼻咽癌细胞株中存在不同程度的Syk基因启动子甲基化状态,5-aza-CdR能有效逆转鼻咽癌细胞株Syk基因启动子的甲基化状态,升高Syk mRNA及蛋白质表达,同时鼻咽癌细胞分化程度越高恢复Syk基因表达的比率越高。     

Epstein-Barr病毒感染对CNE-2Z不同细胞组分中PKC和TPK活性的影响

研究EBV体外再感染CNE-2Z细胞后,不同组分中PKC(蛋白激酶C)和TPK(酪氨酸蛋白激酶)活性的影响,并探讨PKC和TPK活性与细胞增殖的关系。实验分三组即对照组、EBV组和EBV+TPA组,用免疫细胞化学(以小鼠抗EB病毒早期抗原)检测EBV在体外能否再感染CNE-2Z细胞,用特异底物法和特异激活剂法分别测定其PKC和TPK活性,MTT法检测CNE-2Z细胞体外增殖能力。结果显示未处理CNE-2Z细胞中PKC活性为膜性>胞核>胞液,TPK为胞核>膜性>胞液。EBV和EBV+TPA再感染CNE-2Z细胞后,抑制细胞增殖,同时胞液PKC和TPK活性升高,膜性和胞核TPK和膜性PKC活性降低。本研究结果提示,EBV可能通过影响不同细胞组分中PKC和TPK活性来调节CNE-2Z鼻咽癌细胞的增殖。     

人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡的影响

目的:探讨人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡的影响。方法:将生长在对数期的人鼻咽癌CNE-2S细胞分为空白对照组、阴性对照组和实验组。对照组常规培养,阴性对照组采用含有DMSO的培养液培养,实验组在对照组细胞的基础上加入不同浓度人参皂苷单体Rh2处理。采用MTT法测定细胞增殖,PI单染流式细胞术分析各时期细胞所占百分比,Annexin V-PI双染流式细胞仪检测细胞的凋亡情况。结果:与阴性对照组相比,实验组各浓度下的Rh2对CNE-2S细胞均具有显著的增殖抑制作用(P0.05),且随着Rh2浓度的增加而呈现增强的趋势,其中浓度为12.5 mg·L-1 Rh2增值抑制率最低,浓度为100 mg·L-1Rh2增值抑制率最高。不同浓度人参皂苷单体Rh2 G0/G1期细胞分布显著高于阴性对照组(P0.001),且G2/M、S期细胞比例显著低于阴性对照组(P0.01),且随着人参皂苷单体Rh2浓度的增加作用呈现增强的趋势(P0.05);不同浓度的Rh2单体作用24h,CNE-2S细胞早期、晚期凋亡率及总凋亡率均较阴性对照组明显增高(P0.001),并且在Rh2单体浓度为100 mg·L-1时,凋亡率最高。结论:人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡具有显著的影响,并且可能对单体Rh2的浓度存在依懒性。     

Growth inhibition of human nasopharyngeal carcinoma in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies

Monoclonal antibodies (MoAbs) were developed against epidermal growth factor (EGF) receptor on the human epidermoid carcinoma cell line A431. The A431 antigen recognized by the MoAbs has an apparent molecular weight of approximately 170,000, with the same molecular weight as the CNE-2 cell line (poorly differentiated nasopharyngeal carcinoma). Administration of anti-EGF receptor MoAbs inhibited tumor formation, caused by the CNE-2 and A431 cell lines, in athymic mice. When the same MoAbs were used in therapy against Tca8113 (a human tongue carcinoma) and HeLa cells (a human cervical carcinoma), tumor growth was not affected. The number of EGF receptors and the apparent dissociation constants for 125I-EGF on CNE-2 and A431 were 1.3 x 10(5)/cell (Kd 7.7 x 10(-8) M) and 1.4 x 10(6)/cell (Kd 2.4 x 10(-9) M), respectively. Three anti-EGF receptor MoAbs were used in these studies. MoAbs 3 and 176, capable of competing with EGF for receptor binding, showed significant tumor growth inhibition. MoAb 101 was incapable of blocking the binding of EGF to its receptor and was not as effective as MoAbs 3 and 176 in tumor growth inhibition. Our observation is that in vitro, MoAb anti-EGF receptor is cytostatic, rather than cytocidal, against CNE-2 and A431.     

Value of morphometric nuclear image analysis using the Feulgen reaction in renal cell carcinoma

OBJECTIVE: To evaluate retrospectively the ability of morphometric nuclear image analysis to predict survival in patients with renal cell carcinoma. STUDY DESIGN: The subjects were 40 patients with previously untreated renal cell carcinoma. Pathologic stage was determined using Robson's stage system. Nuclear grade was assigned according to the criteria of Fuhrman et al. We used the Feulgen staining technique, which has been widely used for the histochemical assessment of nuclear DNA content. A minimum of 300 nuclei were analyzed from each subject. Five variables in morphometric nuclear image analysis were measured: nuclear area, nuclear perimeter, nuclear ellipticity, nuclear regularity and DNA content. Cox's proportional hazard model was applied to identify prognostic usefulness with respect to survival time. RESULTS: All nuclear morphometric variables but nuclear regularity correlated with tumor grade. According to univariate survival analyses, Robson stage and nuclear ellipticity revealed a prognosis on survival with statistical significance. After adjustments for age and sex, nuclear ellipticity remained the only significant prognostic factor related to survival (P < .01). The survival rates were relatively high for patients with nuclear ellipticity > 773 as compared to those with nuclear ellipticity < 773 (P < .05). CONCLUSION: These findings indicate that morphometric nuclear image analysis using the Feulgen reaction is a reliable and efficient technique and that nuclear ellipticity is the most discriminating morphometric variable for predicting the prognosis of renal cell carcinoma patients.     

MiR-205 determines the radioresistance of human nasopharyngeal carcinoma by directly targeting PTEN

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance severely reduces NPC radiocurability. Here, we have established a radio-resistant NPC cell line, CNE-2R, and investigate the role of miRNAs in radioresistance. The miRNAs microarray assay reveals that miRNAs are differentially expressed between CNE-2R and its parental cell line CNE-2. We find that miR-205 is elevated in CNE-2R. A target prediction algorithm suggests that miR?205 regulates expression of PTE N, a tumor-suppressor. Introducing miR-205 into CNE-2 cells suppresses PTE N protein expression, followed by activation of AKT, increased number of foci formation and reduction of cell apoptosis postirradiation. On the other hand, knocking down miR-205 in CNE-2R cells compromises the inhibition of PTE N and increases cell apoptosis. Significantly, immunohistochemistry studies demonstrate that PTE N is downregulated at late stages of NPC, and that miR-205 is significantly elevated followed the radiotherapy. Our data conclude that miR-205 contributes to radioresistance of NPC by directly targeting PTE N. Both miR-205 and PTE N are potential predictive biomarkers for radiosensitivity of NPC and may serve as targets for achieve successful radiotherapy in NPC.