FasL基因转染诱导类风湿关节炎患者滑膜细胞凋亡的实验研究

Experimental study of apoptosis induced by rhFasL gene transferred into primary cultured synoviocytes from patients with rheumtoid arthritis

目的通过构建FasL重组质粒并转染原代培养的类风湿关节炎(RA)患者滑膜细胞,研究FasL基因对体外培养的RA滑膜细胞的凋亡诱导作用,寻找针对R A关节腔内基因治疗的有效靶基因。方法采用反转录-聚合酶链反应(R T-PCR)方法扩增FasL cD NA全长片段,经Bam HⅠ和XhoⅠ双酶切,将FasL基因导入真核表达载体pcDN A3.1-neo;体外原代培养RA患者及正常人的滑膜细胞;脂质体包裹真核表达质粒pcD NA3.1-FasL及空质粒pcDN A3.1-neo,分别转染处于指数生长期的RA患者及正常人的滑膜细胞;经G418筛选,采用形态学、TUNEL法及AnnexinⅤ/碘化丙啶(PI)染色等方法检测各组滑膜细胞的凋亡情况。结果FasL基因原核及真核重组质粒载体顺利构建,基因序列检测结果与国外报道完全一致;RA患者及正常人的滑膜细胞原代培养、传代顺利完成;RA患者滑膜细胞转染FasL重组质粒(rhFasL)15h后,光镜下可见大量细胞体积缩小、扭曲、变形,胞核中出现颗粒样物质,少数细胞脱落底壁,G418筛选2周后仅有1￣2个/低倍视野细胞存活,且生长相对静止;RA患者滑膜细胞在转染空质粒及正常人滑膜细胞在转染pcDNA3.1-FasL及空质粒后仅有少数细胞变形、反光度下降,很少脱离底壁,G418筛选2￣4周后细胞再次生长并形成克隆;经TUNEL方法检测可见大量转染rhFasL的RA滑膜细胞的?

Objective To observe apoptosis of primary cultured synoviocytes from patients with rheumtoid arthritis (RA) which induced by transfected recombinant plasmid carring Fas ligand (FasL) gene, aimed to detect the aim targets and develope gene therapy for RA by intra-articular injection. Methods FasL cDNA was introducted into the plasmid pcDNA3.1-neo by reverse transcriptase-polymerse chain reaction (RT-PCR) to set up the recombinated human FasL gene expression carrier, Then the pcDNA3.1-FasL was transducted into the primary cultured synovial cells by catholyte liposomes. After screened by G418, transgenic synovial cells were choosen. Finally through the microscope, TUNEL, Annexin Ⅴ-FITC and PI staining with flow cytometry, we observed the apoptosis state of each synovial cell. Results Prokaryotic and eukaryon carrier with FasL gene were established successfilly. The gene sequencing had completely coincident with the lierature. When eukaryon carrier with FasL (pcDNA3.1-FasL) was transducted into the index number livinging RA synovial cells after 15 h, most synovial cells presented distortion transformation, and the minority sheded from the bottom wall. The transgenic RA synovial cells after screened by G418, only a little amount survived about 1~2 per sight, and the cells grew slowly, the normal matched cells were cloned after 2~4 weeks. The FasL gene transgenic RA synovial cells presented positive findings by TUNEL staining. Early apoptosis cells by Annexin Ⅴ/PI staining, but the difference wasn′t significant. Conclusion The eukaryon carrier with FasL (pcDNA3.1-FasL) can be transferred into RA synovial cells. It can induce synovial cell apoptosis.