黄疸患者血清调节人PASMCs增殖及表型转换作用的研究

发布时间：2018-07-02 07:40

本文选题：肺动脉平滑肌细胞 + 黄疸血清　；参考：《第三军医大学》2014年硕士论文

【摘要】：肝肺综合征（hepatopulmonary syndrome，HPS）是一种因肝脏受损引起远端器官肺发生严重病理改变的疾病，以“慢性肝病-肺微血管扩张和增生-低氧血症”为主要特征。在此病理过程中，慢性肝病产生大量的刺激因子（如细胞因子、生长因子、趋化因子等），通过血液循环到达肺脏，作用于肺微血管内皮细胞（pulmonarymicrovascular endothelial cells，PMVECs），导致肺内微血管扩张和增生，使肺部发生氧合障碍出现低氧血症。目前，HPS的发病机制尚不明确。动物实验研究发现大鼠胆总管结扎（common bileduct ligation, CBDL）后肺动脉管壁增厚导致了阻塞性肺血管病变，我们的前期研究发现CBDL大鼠血清可以促进肺动脉平滑肌细胞（pulmonary artery smooth muscle cells,PASMCs）发生表型转换和增殖。此外，大量研究表明PASMCs表型转换和增殖是低氧性肺血管重建的核心变化。这些结果提示低氧环境可能促进HPS大鼠PASMCs发生表型转换和增殖，导致肺动脉管壁增厚，从而加重了肺部低氧血症，，促进了HPS的发生发展。但是，在HPS患者中是否也发生PASMCs表型转换和增殖，尚不清楚，值得进一步研究。而黄疸多见于慢性肝病患者，鉴于此，我们推测：慢性肝病产生大量细胞因子、趋化因子及生长因子释放入血，通过血液循环达到肺脏，作用于PASMCs引起了肺血管的病理性重建，导致了HPS的发生发展，这就是本课题立题的目的。本课题通过原代培养成人PASMCs，观察黄疸患者血清调节人PASMCs增殖及表型转换的变化，同时，检测肝硬化患者血清中细胞因子的变化情况，筛选出起关键作用的细胞因子，从而为HPS的防治提供新的靶点。研究内容： 1.应用含适量的PDGF的高糖培养基原代培养成人PASMCs，运用光镜、免疫荧光染色法进行鉴定。 2.黄疸患者血清的收集，分别用正常和黄疸血清刺激人PASMCs，用CCK-8分别检测其增殖情况，Western blot检测其calponin和α-SM-actin蛋白的变化情况。 3.应用luminex-XMAP液相芯片检测肝硬化患者血清中61种细胞因子的变化情况研究结果： 1.应用改良的成人PASMCs培养方法进行原代培养，细胞在光镜下呈梭形，细胞之间突起相互接触，可见典型的“峰-谷”征象，细胞内calponin和α-SM-actin蛋白经免疫荧光染色鉴定均为阳性，说明了人PASMCs培养成功。 2.黄疸患者血清促进人PASMCs的增殖。与正常血清刺激人PASMCs比较，黄疸血清刺激人PASMCs的体外增殖能力明显增强。在不同时间段内，48h内的增殖能力最强，虽然48h到72h增殖能力较弱，但仍比24h内增殖能力强。说明了黄疸患者血清可以促进人PASMCs在体外增殖。 3.黄疸患者血清促进人PASMCs发生表型转换作用24h后，western bolt检测结果发现，与正常血清相比，黄疸血清刺激α-SM-actin蛋白变化不明显，而calponin蛋白却表达升高。但是，随着作用时间延长，48h和72h后，黄疸血清刺激α-SM-actin和calpnion蛋白的表达均明显减少。 4. luminex-XMAP液相芯片检测结果显示61种细胞因子中，IL-6，IL-8，IL-15，IL-16，IL-23，TNF-α，IL-28A，LIF，I-309，IP10，MCP-4，6Ckine，BCA-1，MIP-1δ，INF-γ表达明显升高，而其他的细胞因子（如IL-3、IL-7、TRAC、TRAIL、GRO、MCP-1、MCP-2、ENA-78及sCD40L）表达明显下降。结论： 1.改良的成人PASMCs培养方法克服了成人肺动脉平滑肌细胞培养困难的问题，同时将人PASMCs应用于实验更符合人体形态学及生物学特征。 2.黄疸患者血清促进人PASMCs体外发生表型转换和增殖。 3.通过luminex-XMAP液相芯片技术筛选出肝硬化患者血清中的变化较为明显的细胞因子，为进一步研究肝源性肺疾病机制提供科学依据。
[Abstract]:In this pathological process , chronic liver disease produces a large amount of stimulating factor ( such as cytokines , growth factors , chemokine , etc . ) , and reaches the lungs through the circulation of blood .

In this study , we have found that chronic liver disease can promote the phenotype conversion and proliferation of pulmonary artery smooth muscle cells ( PASI ) . In addition , it is suggested that the changes of pulmonary vascular wall thickness caused by chronic liver disease can promote the alteration and proliferation of pulmonary vascular smooth muscle cells ( PASI ) .

Study Content :

1 . A high - sugar culture medium containing a proper amount of PDGF was used to culture adult PASI , and was identified by light microscope and immunofluorescence staining .

2 . The serum of patients with jaundice was collected and stimulated with normal and jaunicteric serum to stimulate human PASI . CCK - 8 was used to detect their proliferation . Western blot was used to detect the changes of calpain and 伪 - SM - actin .

3 . Changes of 61 cytokines in serum of patients with liver cirrhosis using luminex - XMAP liquid chip

Results of the study :

1 . Primary culture was carried out by using the improved culture method of PASI . The cells were fusiform under the light microscope and the cells were in contact with each other . The typical " peak - valley " signs were observed , and the calpain and 伪 - SM - actin protein in the cells were identified as positive by immunofluorescence staining .

2 . In the patients with jaundice , the proliferation of PASI was promoted . Compared with the normal serum - stimulated human PASI , the proliferative ability of the human PASI stimulated by jaundice was strongest . Although the proliferative ability was weak within 48 h and 72 h after 48 h , the proliferative ability was stronger than that in 24 h . The serum of patients with jaundice could promote the proliferation of human PASI in vitro .

3 . After 24 h , the serum stimulated 伪 - SM - actin was not obvious , but calpain protein was increased . However , the expression of 伪 - SM - actin and calphedrin was significantly decreased after 48 h and 72 h , as compared with normal serum .

4 . The expression of IL - 6 , IL - 8 , IL - 15 , IL - 16 , IL - 23 , TNF - 伪 , IL - 28A , LIF , I - 309 , IL - 15 , IL - 28A , LIF , I - 309 , MIP - 1伪 , IL - 28A , LIF , I - 309 , MIP - 1伪 , INF - 纬 were significantly increased in 61 cytokines , while other cytokines ( such as IL - 3 , IL - 7 , TRAC , TRAIL , GRO , MCP - 1 , MCP - 2 , IL - 78 and sCD40L ) decreased significantly .

Conclusion :

1 . The improved method of adult PASI culture overcomes the difficult problem of adult pulmonary artery smooth muscle cell culture , and meanwhile , it can be applied to the experiment to be more consistent with human morphology and biological characteristics .

2 . In the serum of patients with jaundice , the phenotype conversion and proliferation of human PASI were promoted in vitro .

3 . The cytokines in serum of patients with liver cirrhosis were screened by luminex - XMAP liquid - phase chip technique , which provided scientific basis for further study on the mechanism of hepatic origin lung disease .
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

【参考文献】