Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by In Situ Hybridization

Objective：Laryngeal squamous cell carcinoma（LSCC） is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol abuse.Human papilloma virus（HPV） is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including LSCC.This retrospective study was to investigate the prevalence of HPV infection in patients with LSCC.Methods：In situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6 mRNA.Results：The positive rate of HPV16 E6 mRNA was 36.36%（36/99） in laryngeal squamous cell carcinoma（LSCC）,whereas only 3 of 50（6%） specimens of the normal laryngeal mucosa as a control group showed positive results（P0.05）.Additionally,there was no corelation between HPV16 and age,gender,clinical stage,nodal status and tumor site（P0.05）.Conclusion：The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types（especially type 16） were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell carcinoma.However,their clinical value remains to be further investigated.

Cytotoxicity of Modified Nonequilibrium Plasma with Chlorhexidine Digluconate on Primary Cultured Human Gingival Fibroblasts

The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate（CHX） on human gingival fibroblasts（HGFs）, and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3–7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min（control group）, 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8（CCK-8） assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group（P〉0.05）. However, there were significant differences between all the other treated groups and the control group（P〈0.05）. When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.

Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis

Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli （UPEC） to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium （K-SFM） with epidermal growth factor （EGF） and bovine pituitary extract （BPE）. Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678- 54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnfl of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.

Relation between the Expression of K-ras in Hep-2 Cells and Development of Laryngeal Carcinoma~*

Objective： To investigate the expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and its significance for establishing a solid foundation for further study of the relationship between human laryngeal squamous cell carcinoma and K-ras gene point mutations. Methods： The expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and human pancreatic carcinoma cell lines （MIAPaCa-2） was detected by using RT-PCR. Results： The expression of K-ras mRNA in Hep-2 and MIAPaCa-2 was strong and positive. Conclusion： The expression of K-ras mRNA in human laryngeal squamous cell carcinoma cell lines （Hep-2） is positive. Development of laryngeal carcinoma might be related to the activation of K-ras gene point mutation.

Three-dimensional collagen-based scaffold model to study the microenvironment and drug-resistance mechanisms of oropharyngeal squamous cell carcinomas

Objective:Squamous cell carcinoma(SCC)represents the most common histotype of all head and neck malignancies and includes oropharyngeal squamous cell carcinoma(OSCC),a tumor associated with different clinical outcomes and linked to human papilloma virus(HPV)status.Translational research has few available in vitro models with which to study the different pathophysiological behavior of OSCCs.The present study proposes a 3-dimensional(3 D)biomimetic collagen-based scaffold to mimic the tumor microenvironment and the crosstalk between the extracellular matrix(ECM)and cancer cells.Methods:We compared the phenotypic and genetic features of HPV-positive and HPV-negative OSCC cell lines cultured on common monolayer supports and on scaffolds.We also explored cancer cell adaptation to the 3 D microenvironment and its impact on the efficacy of drugs tested on cell lines and primary cultures.Results:HPV-positive and HPV-negative cell lines were successfully grown in the 3 D model and displayed different collagen fiber organization.The 3 D cultures induced an increased expression of markers related to epithelial–mesenchymal transition(EMT)and to matrix interactions and showed different migration behavior,as confirmed by zebrafish embryo xenografts.The expression of hypoxia-inducible factor 1α(1α)and glycolysis markers were indicative of the development of a hypoxic microenvironment inside the scaffold area.Furthermore,the 3 D cultures activated drug-resistance signaling pathways in both cell lines and primary cultures.Conclusions:Our results suggest that collagen-based scaffolds could be a suitable model for the reproduction of the pathophysiological features of OSCCs.Moreover,3 D architecture appears capable of inducing drug-resistance processes that can be studied to better our understanding of the different clinical outcomes of HPV-positive and HPV-negative patients with OSCCs.

Studies on the interrelationship of Chinese laryngeal carcinoma and human papillomavirus

The studies described herein emphasize HPVDNA detection by applying consensus primers and multiple primers of modified polymerase chain reaction (PCR) to 124 cases of fresh tissue samples with different lesions of larynx. The consensus primers used were able to detect 9 types of HPVsDNAs ( HPV 6 , 11 , 16 , 18, 31 , 33,35, 42, and 583 , from which the positive cases were picked out for further identification of their genometypes of HPV-DNA by using multiple primers PCR. The results areas follows : (1)In the group of laryngeal carcinoma,the total positive rate of HPV infection was 49. 1 %(28/57) : 15. 8%(9/57)for HPV18, 12. 3%(7/57)for HPV16,5. 3 %(3/57) dual infection for HPV16 and HPV18 , 3. 5%(2/57) for mixed infection of HPV6/11 and HPV18 , and 12. 3%(7/57)for the other types. (2) In the cervical metastatic lymphnode group, 3 of the 14 cases (21. 4%) of cervical metastatic lymphnode showed positive HPV, among which there was 1 case of HPV16 infection , 1 case of HPV18 infection ,and 1 case of HPV16 and HPV18 dual infection, resulting in a rate at 7. 1% for the respective cases immediately above. (3) In the pecarcinomatous lesion group ,the positive rate of HPV infection tmixed infection of HPV6/11 and HPV18) was 11. 1 %(1/9). (4)In the vocal cord polypus group , the rate of positive reaction (HPV6/11) was 7.1% (1/14). (5) In the nomal laryngeal tissue group , 15 cases of normal laryngeal tissue adjacent to the carcinoma and 15 cases of normal laryngeal tissue opposite to the carcinoma were HPV-DNA negative.The results showed that the occurrence and development of laryngeal carcinoma were closely related to HPV infection. The distribution of genometypes of HPV varied in different lesions of larynx. The carcinogenic action of HPV in laryngeal carcinoma is also discussed in this paper.

原代培养人喉鳞癌细胞中SIX1、TGF-β、VEGF-C表达的相关性

目的探讨原代人喉鳞癌细胞中SIX1、TGF-β、VEGF-C表达相关性。方法取新鲜人喉鳞状细胞癌组织,进行原代细胞培养,利用基因干扰技术,制备SIX1、TGF-β及SIX1+TGF-β靶向si RNA转染至人喉鳞癌细胞,转染成功后将实验细胞分为5组:A组(未转染组)、B组(转染空载体组)、C组(SIX1-si RNA组)、D组(TGFβ-si RNA组)、E组(SIX1+TGF-β-si RNA组)。Western blot及细胞爬片免疫荧光检测各组细胞SIX1、TGF-β及VEGF-C蛋白的表达。RT-PCR检测各组细胞SIX1、TGF-β及VEGF-C m RNA的表达。结果 SIX1、TGF-β、VEGF-C蛋白及其m RNA的表达在未转染组、转染空载体组中差异无统计学意义。同未转染组相比,SIX1蛋白及其m RNA的表达在SIX1-si RNA组降低的同时出现了VEGF-C表达的降低;TGF-β蛋白及其m RNA的表达在TGFβ-si RNA组降低的同时也出现了VEGF-C表达的降低,差异均有统计学意义。同SIX1-si RNA组、TGFβ-si RNA组相比,VEGF-C蛋白及其m RNA的表达在SIX1+TGF-β-si RNA组未出现进一步降低。结论 SIX1和TGF-β能够共同作用促进VEGF-C蛋白及其m RNA的表达,进而促进人喉鳞癌的淋巴转移。SIX1、TGF-β的变化可能成为临床抑制人喉鳞癌淋巴结转移的潜在靶点。

人皮肤成纤维细胞原代培养及生物学特性

目的探索和建立人皮肤成纤维细胞原代培养的方法,为皮肤成纤维细胞在临床医学中的应用提供可靠的科学依据。方法胰蛋白酶消化法分离培养人皮肤成纤维细胞;细胞生长曲线及MTT法测定细胞增殖能力;流式细胞仪测定细胞生长周期及细胞增殖指数;倒置显微镜下观察成纤维细胞生长状态;HE染色观察细胞爬片下的形态及着色;免疫组织化学染色观察成纤维细胞中间丝波形蛋白;电子显微镜下观察成纤维细胞超微结构。结果体外用胰蛋白酶消化法建立了人皮肤成纤维细胞;传5代内细胞及传5代内冻存复苏后人皮肤成纤维细胞增殖能力均很强;倒置镜下观察、HE染色、波形蛋白免疫组化染色及电镜下观察鉴定培养细胞的形态及生物学特性符合成纤维细胞。结论本研究确立了体外人皮肤成纤维细胞原代培养。

短发夹RNA沉默hTERT基因对人喉癌裸鼠移植瘤的生长抑制作用

背景与目的:RNA干扰(RNAinterference,RNAi)是指双链RNA导入细胞后诱导靶mRNA发生特异性的降解,导致基因转录后沉默的现象。RNAi在基因功能和抗病毒基因治疗等方面均有报道。但对喉癌等头颈恶性肿瘤中高表达的人端粒酶逆转录酶(humantelomerasereversetranscriptase,hTERT)基因,目前尚未见报道。本课题利用RNAi技术,研究短发夹RNA(shorthairpinRNA,shRNA)抑制hTERT基因表达对裸鼠喉鳞状细胞癌皮下移植模型的抑瘤作用。方法:根据hTERTcDNA序列构建表达hTERTmRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA。建立人喉癌Hep-2细胞株裸鼠皮下接种模型。将pshRNA转染入荷瘤裸鼠瘤体内,观察肿瘤生长情况。以激光共聚焦显微镜观察质粒在瘤组织内的表达;以HE染色法观察质粒治疗后瘤组织的病理改变;以原位末端标记法(TUNEL法)检测肿瘤细胞的凋亡情况;以免疫组化SP法检测hTERT蛋白在肿瘤内的表达;光镜下观察心、肝、肾、脾结构的改变并测量血液学和血液生化指标。结果:pshRNA组(A组)、空质粒载体组(B组)与生理盐水组(C组)之间比较,A组与C组瘤体积有显著性差异(P<0.01),B组与C组之间瘤体积无显著性差异(P>0.05),抑瘤率为76.50%。pshRNA及空质粒载体转染入瘤体后,共聚焦显微镜下见大量的癌细胞表达绿色荧光。病理学检查及TUNEL检测发现:A组肿瘤生长受到抑制,细胞分裂相少见,可见大量肿瘤细胞坏死及凋亡,该组肿瘤细胞的凋亡指数[(26.47±4.25)%]明显高于B组[(3.40±1.41)%]和C组[(2.73±1.35)%]。给予pshRNA后瘤体内hTERT蛋白表达明显下调,而且对心、肝、肾、脾无明显损害,对造血系统无影响。结论:表达shRNA的DNA载体质粒沉默hTERT基因可显著抑制人喉癌裸鼠肿瘤的生长,而且对心、肝、肾、脾及造血系统无明显的毒副作用。

体外原代培养人视网膜色素上皮细胞(英文)

目的:探讨建立体外原代培养人类视网膜色素上皮细胞(RPE)技术。为研究溶血磷脂酸(lysophos-phatidic acid, LPA)对培养的人类视网膜色素上皮细胞DNA合成与增殖的作用。方法:取自愿者贡献的意外事故成年眼球,用2.5g/L胰酶消化获取人RPE细胞、150mL/L胎牛血清的DMEM培养液培养,细胞接近融合状态时进行传代培养。取自愿者贡献的眼球并游离其视网膜色素上皮细胞,用DMEM加100mL/L血清及MEM氨基酸和庆大霉素,进行细胞传代培养。用溶血磷脂酸、转移因子β2(TGF-β2)及LPA+TGF-β2进行视网膜色素上皮细胞增殖试验,用细胞染色法进行细胞计数,提取视网膜色素上皮细胞DNA,用Spectrophotometer进行DNA定量测定。结果:RPE原代细胞镜下为圆形,大小不一,内含较多的色素颗粒,胞核无法辨认。原代细胞培养贴壁后3d增殖速度明显加快,至4～5d即可基本融合,细胞浆内色素颗粒则随传代次数增多而逐渐减少。第3代培养的视网膜色素上皮细胞膜表面可见微绒毛,细胞质内细胞器丰富,线粒体量多,体积较小,内外膜分界清晰,嵴较短。色素颗粒散在分布于胞浆内,多数细胞质内数量较少呈高电子密度包含物。LPA对原代培养的视网膜色素上皮细胞增殖有促进作用。含有和/或缺乏10mL/L小牛血清的两种LPA(10μmol/L雪均明显刺激视网膜色素上皮细胞?

人肾近端小管上皮细胞的原代培养、传代及鉴定方法研究

目的:创建一种重复性好、细胞量多的人肾近端小管细胞(PTC)培养方法.方法:取新鲜正常人肾组织分离纯化,用含10%新生牛血清等营养充分的DMEM/F12(1:1)液进行近端小管细胞的原代培养及传代,并以免疫组化、酶化学染色、透射电镜鉴定.结果:培养5～6天后融合成单层细胞,形态呈鹅卵石样,可传代7～9代,鉴定确定为人肾近端小管细胞,重复培养10次,均能稳定获得同样细胞,每克肾皮质可分离培养出(6～12)×106个PTC.结论:此培养方法可在7天内获得人肾近端小管细胞,且重复性好,细胞数量多,为进行肾小管细胞治疗提供必需的细胞,也为研究肾小管病变提供实验平台.

胰蛋白酶消化法和组织块法原代培养人包皮成纤维细胞的比较

小鼠胚胎成纤维细胞(MEFs)是目前国际上公认的人胚胎干细胞(hESCs)体外培养的饲养层细胞,但MEFs存在寿命短、动物源性污染等问题,需要探索适于hESCs体外培养且寿命长的人源性饲养层.采用胰蛋白酶消化法和组织块法分别原代培养人包皮成纤维细胞(hFFs),探索hFFs原代培养的最佳方法,为hFFs作为饲养层在hESCs研究中的应用提供可靠的科学依据;通过倒置显微镜下观察其生长状态和免疫细胞化学染色鉴定,结果显示两种原代培养方法获得的hFFs的形态及生物学特性均符合成纤维细胞;通过测定细胞生长曲线及MTT法检测,结果显示两种原代培养方法获得的不同代数的hFFs均具有较高的增殖活性,传代10余代仍能保持较好的细胞形态,可以制备成饲养层用于hESCs的研究.

影响喉癌组织原代培养成功率因素的研究

目的研究不同组织分离方法、肿瘤TNM分期、细胞分化程度等因素对喉癌组织原代培养细胞生长率的影响。方法对17例喉癌组织分别应用组织块、胶原酶和机械分离法进行原代培养,观察以上3种组织分离法、肿瘤细胞分化程度以及TNM分期对喉癌原代培养细胞生长情况的影响,并用统计学分析其结果。结果组织块、胶原酶及机械分离法的细胞生长率分别为64.7%、58.8%和11.8%,其中组织块法和胶原酶法比较差异无统计学意义(P=1.000),而织块法和胶原酶法与机械分离法比较差异均有统计学意义(P=0.004和0.008)。鳞癌细胞高分化组细胞生长率为33.3%,中/低分化组为90.9%,2组比较差异有统计学意义(P=0.028)。结论细胞培养的生长率与肿瘤组织的分化程度和组织分离方法有关。在喉癌的原代培养中应尽量选取细胞分化程度差的标本并且选用组织块分离法进行培养。

HPV16/18-E6、p53和MDM2蛋白在人喉鳞癌中的表达及相关性研究

目的：探讨HPV16／18-E6、p53和MDM2蛋白在人喉鳞状细胞癌（鳞癌）组织中的表达及其在喉鳞癌发生和发展过程中的相互关系。方法：用EnVision免疫组织化学法检测72例喉鳞癌、24例喉癌前病变、15例声带息肉和8例癌周正常喉组织标本中HPV16／18-E6、p53和MDM2蛋白的表达并比较，对喉鳞癌与临床病理因素进行相关性分析。、结果：癌周正常喉组织、声带息肉、喉癌前病变和喉鳞癌组织中HPV16／18-E6蛋白的阳性表达率分别为0（0／8）、6．7％（1／15）、12．5％（3／24）和37．5％（27／72），p53蛋白的阳性表达率分别为0（0／8）、13．3％（2／15）、25．0％（6／24）和52．8％（38／72），MDM2蛋白的阳性表达率分别为0（0／8）、6．7％（1／15）、37．5％（9／24）和70．8％（51／72），HPV16／18-E6、p53和MDM2蛋白的阳性表达在声带息肉、喉癌前病变与喉鳞癌组之间均有显著性差异（P〈0．05）。HPV16／18-E6蛋白和MDM2蛋白的阳性表达在病理Ⅰ～Ⅲ级肿瘤之间呈明显升高趋势，有显著性差异（P〈0．05）。HPV16／18E6蛋白的阳性表达与临床分期和淋巴结转移明显相关（P〈0．05）。p53蛋白阳性表达与HPV16／18-E6蛋白、MDM2蛋白阳性表达有显著正相关性（P〈0．05）。结论：HPV16／18-E6、MDM2蛋白的过表达和p53的失活与人喉鳞癌的发生发展密切相关。

人嗜铬细胞瘤细胞的原代培养及鉴定

本研究拟建立人嗜铬细胞瘤细胞的原代培养方法。采用连续分次胶原酶消化法分离培养人嗜铬细胞瘤细胞 ,采用细胞培养液中儿茶酚胺水平检测、多聚甲醛诱发荧光及细胞嗜铬粒蛋白A (CgA)和神经元特异性烯醇化酶(NSE)的免疫组化染色等方法进行细胞性质和功能鉴定 ,并用噻唑兰 (MTT)法观察原代培养的人嗜铬细胞瘤细胞的生长状况。结果表明 ,人嗜铬细胞瘤细胞在培养 3～ 7天时生长较快 ,7天后细胞开始分化。经检测细胞培养液中的儿茶酚胺浓度、多聚甲醛诱发荧光等 ,证明该细胞有合成和分泌儿茶酚胺的功能。并且培养的细胞CgA和NSE免疫组化染色阳性。因此 ,本研究成功建立了人嗜铬细胞瘤细胞的原代培养方法 ,并鉴定其具有嗜铬细胞瘤的分泌和表达功能 。

人参皂苷Rg3对原代人喉鳞癌细胞SIX1、TGF-β、VEGF-C表达的影响

目的:探讨人参皂苷Rg3对原代培养人喉鳞癌细胞中SIX1、TGF-β、VEGF-C表达的影响。方法:培养原代人喉鳞癌细胞,待细胞稳定传代后将其分为3组:A组(正常对照组)、B组(顺铂对照组)、C组(Rg3处理组)。Westernblot及细胞爬片免疫荧光检测各组细胞SIX1、TGF-β及VEGF-C蛋白的表达。RT-PCR检测各组细胞SIX1、TGF-β及VEGF-C mRNA的表达。结果:正常对照组细胞SIX1、TGF-β及VEGF-C蛋白及其mRNA表达均呈现强阳性。同正常对照组相比,顺铂对照组及Rg3处理组细胞TGF-β表达无明显变化,SIX1及VEGF-C蛋白及其mRNA表达下调,差异有统计学意义;同顺铂对照组相比,Rg3处理组细胞TGF-β表达无明显变化,SIX1及VEGF-C蛋白及mRNA表达出现了明显下调,差异有统计学意义。结论:Rg3能够通过降低人喉鳞癌细胞SIX1表达下调VEGF-C蛋白表达,进一步抑制喉鳞癌淋巴转移。同时提示,Rg3有比顺铂更强的抑制喉鳞癌淋巴转移的作用。

人脐带间充质干细胞的原代培养及多向分化潜能的研究

目的探讨人脐带间充质干细胞（hUCMSC）的体外分离培养鉴定方法及多向分化潜能。方法应用Ⅱ型胶原酶和透明质酸酶联合消化法及组织贴块培养法对hUCMSC进行体外培养。在倒置显微镜下观察不同方法获得的hUCMSC的生长特点；台盼蓝法测定细胞传代成活率；采用生长曲线、MTT法测定hUCMSC的增殖、流式细胞术（FCM）测定细胞周期变化及细胞免疫表型变化；采用相应试剂盒鉴定其向成脂细胞、成骨细胞的分化，采用免疫荧光细胞化学技术检测向成心肌样细胞的分化、采用荧光标记技术检测hUCMSC向血管内皮细胞的分化。结果酶消化法分离培养的细胞1d后，在倒置相差显微镜下可见细胞贴壁呈圆形生长，4d后生长迅速呈长梭形，7d后由中心向周围生长且增殖明显，10d后达80％融合即可传代；贴块法培养的细胞7d后，可见细胞从组织块边缘长出，10d后细胞数量逐渐增多，16d后细胞生长迅速呈单层致密排列即可传代，细胞传代成活率均为96％以上。2种方法获得的第3代细胞生长曲线近似“S”形、MTr法显示3～5d细胞增殖较明显；酶消化法培养的细胞G0／G1期和s＋G2／M期所占比例分别为88．78％和10．21％，组织贴块法培养的细胞G0／G1期和S＋G2／M期所占比例分别为84．82％和13．87％，细胞DNA周期无明显差异；2种方法获得的细胞经FCM检测CD90、CD105、CD73阳性率均为99％以上为高表达；CD45、CD34、CD14、CD11b、CD79a、CD19、HLA—DR低表达；2种方法获得的细胞在体外诱导都能向成骨细胞、成脂细胞、成心肌样细胞及血管内皮细胞分化，其诱导阳性率均为90％以上。结论酶消化法和贴块法均可高效获得hUCMSC，与贴块法培养相比较，酶消化法能更有效的获得扩增迅速、细胞成分单一的细胞。

多胺生物合成抑制剂对Hep-2细胞生长及端粒酶活性影响的研究

目的 观察多胺生物合成抑制剂二氟甲基鸟氨酸 (difluoromethylornithine,DFMO)对人喉鳞状细胞癌细胞Hep 2生长特性及端粒酶活性的影响 ,以期为抑制多胺生物合成逆转肿瘤恶性表型的分子机理寻找线索。方法 根据细胞形态、细胞生长曲线及流式细胞计 (flowcytometer)观察分析Hep 2细胞生长状况 ;用端粒酶重复序列扩增法 (telomeraserepeatamplificationprotocol,TRAP)检测Hep 2细胞端粒酶活性。结果 DFMO (2 .5mmol/L或 5mmol/L ,5d)处理Hep 2细胞 ,其生长受到明显抑制 ;G1期细胞增多 ,而S期细胞减少 ;细胞形态及FCM证明有细胞凋亡诱导 ;端粒酶活性受到明显抑制。当用腐胺与DFMO同时处理Hep 2细胞时可防止上述变化的发生。结论 多胺生物合成抑制剂可引起Hep 2细胞的增殖抑制及凋亡诱导 ,这些变化与端粒酶活性的抑制有关。

原代培养人脐动脉内皮细胞的生长与增殖

在未用贴附基质和生长因子情况下,成功地培养了人脐动脉内皮细胞(human umbilicalartery endothelial cell,HUAEC)。接种密度为1×10~5/cm^2。采用HE染色、显微计量法及透射电镜术研究了内皮细胞的形态、生长行为与增殖。(1)接种后24h已出现岛状细胞团;48h细胞团增大,可区分出细胞密集的中央区及细胞密度较小的周边区;72h大部分细胞团已汇合;216h内皮细胞衰退、脱落。(2)HUAEC呈较长的多边形,相邻细胞间以短突相连,内胞质呈颗粒状弱嗜碱性,外胞质扁薄。(3)一个细胞团所含平均细胞数,24h为17.39±3.79个;48h为131.14±22.83个,72h为454.16±111.39个,表明在接种后72h内细胞倍增9次。(4)在细胞呈指数生长的24～72h时出现许多无丝分裂像,但未见有丝分裂像;至120h细胞生长缓慢时才出现有丝分裂像。(5)电镜下可见内皮细胞含Weibel-Palade小体。

内毒素对牙龈成纤维细胞增殖活性及分泌基质金属蛋白酶的影响

目的观察内毒素对牙龈成纤维细胞增殖活性及分泌基质金属蛋白酶-1,3量的影响,探讨基质金属蛋白酶在牙周炎致病机制中的可能作用。方法通过改良酶消化组织块法进行牙龈成纤维细胞的原代培养,用内毒素对其进行刺激,运用MTT法观察内毒素对牙龈成纤维细胞增殖活性的影响,通过免疫组化法检测内毒素对牙龈成纤维细胞分泌基质金属蛋白酶-1,3的影响。结果MTT结果显示,低浓度内毒素在短期内可以促进牙龈成纤维细胞的增殖,但随着时间的延长,也表现为对细胞的毒性作用,抑制其增殖;高浓度内毒素抑制牙龈成纤维细胞的增殖;免疫组化研究结果表明,未受LPS刺激的牙龈成纤维细胞MMP-1,3表达弱阳性,受刺激后阳性表达增强,在12h时达到顶峰,且MMP-3的整体表达较MMP-1弱。结论LPS可以影响牙龈成纤维细胞的增殖活性;LPS可以促进牙龈成纤维细胞对基质金属蛋白酶的分泌,加快牙周细胞外基质的降解,加速牙周炎的进程。