摘要
目的:观察正常人近端肾小管上皮细胞(HK2)是否表达黏附分子CD146,初步探讨CD146与肾小管上皮细胞的关系及其生理意义。方法:实验于2005-05/2006-02在上海交通大学医学院临床检验系实验室完成。①实验材料:人近端肾小管上皮细胞株(HK2,由中国科学院上海生物化学与细胞生物学研究所惠赠)。②实验干预:体外培养的HK2连续观察72h。③实验评估:采用倒置显微镜、光学显微镜下观察肾小管上皮细胞的形态;用反转录-聚合酶链反应方法检测CD146 mRNA的表达;流式细胞仪和免疫荧光法测定CD146蛋白质的表达及其定位;进一步在培养细胞的上清液中检测CD146的可溶性形式(sCD146)。结果:①肾小管上皮细胞的形态学观察和鉴定:传代培养的HK2三四天融合后呈铺路石样铺于培养瓶底,相关抗原检测抗人keratin阳性,抗Ⅷ因子相关抗原阴性。②CD146 mRNA水平的表达:HK2在培养早期(24h)即表达CD146 mRNA(0.092±0.012),但延长细胞的培养时间似乎并没有进一步改变CD146 mRNA的表达水平(0.097±0.005,0.113±0.015,P>0.05)。③CD146蛋白质水平的表达和定位:CD146不仅位于肾小管上皮细胞膜上,而且在细胞核和胞浆中也有表达。体外培养的HK2相互融合时,位于细胞膜上的CD146表达增强,呈线性、持续性地表达于细胞-细胞间的连接部位,同时胞内的CD146标记也相应增强。④细胞上清液中sCD146的检测:HK2上清液中存在CD146的可溶性形式(sCD146)。培养细胞观察24~72h,sCD146水平在48h升高[(18.00±0.80)μg/L],到72h达到高峰[(29.33±1.22)μg/L],与24h[(13.87±0.46)μg/L]比较,差异均有显著性意义(P<0.05)。结论:人近端肾小管上皮细胞组成性地表达CD146,是肾小管上皮细胞新的生物学标志;CD146在细胞内外的表达强度有赖于细胞间联系的建立和细胞增殖的程度,CD146在促进细胞生长和维持组织完整性等方面发挥重要作用。肾小管上皮细胞上清液�
AIM: To observe whether CD146 is expressed on human renal tubular epithelial cells (TECs) (HK2) or not, and to make further discussion on the possible physiological meaning and relationship between CD146 and TECs. METHODS: The experiment was performed at the Laboratory of Department of Clinical Laboratory, Medical College, Shanghai Jiao Tong University from May 2005 to February 2006. (1)Human TECs (HK2) was presented by Shanghai Institute of Biochemistry and Cytobiology of Chinese Academy of Sciences. (2) HK2 cultured in vitro was observed successively for 72 hours. (3)Cell morphology of TECs was observed under inverted microscope and optical microscope. Expression of CD146 mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), while the expression of CD146 was assessed and located by flow cytometry and immunofluorescence method. A soluble form of CD146 (sCD146) was detected in the supernatant of cultured HK2 media. RESULTS: (1)Observation and identification of TECs morphology: 3-4 days after confluence, HK2 spread at the bottom of culture flask and arranged in shape of typical "cobble-stone". Related antigen showed anti-human keratin was positive and anti-Ⅷ factor related antigen was negative. (2)Expression of CD146mRNA: HK2 CD146 mRNA was detected at hour 24 in culture (0.092±0.012), but the prolongation of cell culture did not further change the expression of CD146 mRNA (0.097±0.005,0.113±0.015,P〉 0.05). (3)Expression and location of CD146 protein: CD146 was constitutively expressed at the HK2 cell membrane, and in the cytoplasm or nucleus as well. CD146 was increasingly concentrated on cell-cell conjunction when the cells reached confluence in vitro and showed linearity and continuity. At the same time, CD146 labeled in cells was also strengthened. (4)Detection of sCD146 in supernatant: A soluble form of CD146 (sCD146) was also detected in the HK2 supernatant liquid. The sCD146 level was continuously
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第27期5364-5368,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
上海市科学自然基金资助(05ZR14086)
上海市医苑新星(02xx46)~~