發(fā)布時間：2018-07-02 07:40

  本文選題：肺動脈平滑肌細胞 + 黃疸血清　； 參考：《第三軍醫(yī)大學》2014年碩士論文

【摘要】：肝肺綜合征（hepatopulmonary syndrome，HPS）是一種因肝臟受損引起遠端器官肺發(fā)生嚴重病理改變的疾病，以“慢性肝病-肺微血管擴張和增生-低氧血癥”為主要特征。在此病理過程中，慢性肝病產(chǎn)生大量的刺激因子（如細胞因子、生長因子、趨化因子等），通過血液循環(huán)到達肺臟，作用于肺微血管內(nèi)皮細胞（pulmonarymicrovascular endothelial cells，PMVECs），導致肺內(nèi)微血管擴張和增生，使肺部發(fā)生氧合障礙出現(xiàn)低氧血癥。 目前，HPS的發(fā)病機制尚不明確。動物實驗研究發(fā)現(xiàn)大鼠膽總管結扎（common bileduct ligation, CBDL）后肺動脈管壁增厚導致了阻塞性肺血管病變，我們的前期研究發(fā)現(xiàn)CBDL大鼠血清可以促進肺動脈平滑肌細胞（pulmonary artery smooth muscle cells,PASMCs）發(fā)生表型轉(zhuǎn)換和增殖。此外，大量研究表明PASMCs表型轉(zhuǎn)換和增殖是低氧性肺血管重建的核心變化。這些結果提示低氧環(huán)境可能促進HPS大鼠PASMCs發(fā)生表型轉(zhuǎn)換和增殖，導致肺動脈管壁增厚，從而加重了肺部低氧血癥，，促進了HPS的發(fā)生發(fā)展。但是，在HPS患者中是否也發(fā)生PASMCs表型轉(zhuǎn)換和增殖，尚不清楚，值得進一步研究。而黃疸多見于慢性肝病患者，鑒于此，我們推測：慢性肝病產(chǎn)生大量細胞因子、趨化因子及生長因子釋放入血，通過血液循環(huán)達到肺臟，作用于PASMCs引起了肺血管的病理性重建，導致了HPS的發(fā)生發(fā)展，這就是本課題立題的目的。本課題通過原代培養(yǎng)成人PASMCs，觀察黃疸患者血清調(diào)節(jié)人PASMCs增殖及表型轉(zhuǎn)換的變化，同時，檢測肝硬化患者血清中細胞因子的變化情況，篩選出起關鍵作用的細胞因子，從而為HPS的防治提供新的靶點。 研究內(nèi)容： 1.應用含適量的PDGF的高糖培養(yǎng)基原代培養(yǎng)成人PASMCs，運用光鏡、免疫熒光染色法進行鑒定。 2.黃疸患者血清的收集，分別用正常和黃疸血清刺激人PASMCs，用CCK-8分別檢測其增殖情況，Western blot檢測其calponin和α-SM-actin蛋白的變化情況。 3.應用luminex-XMAP液相芯片檢測肝硬化患者血清中61種細胞因子的變化情況 研究結果： 1.應用改良的成人PASMCs培養(yǎng)方法進行原代培養(yǎng)，細胞在光鏡下呈梭形，細胞之間突起相互接觸，可見典型的“峰-谷”征象，細胞內(nèi)calponin和α-SM-actin蛋白經(jīng)免疫熒光染色鑒定均為陽性，說明了人PASMCs培養(yǎng)成功。 2.黃疸患者血清促進人PASMCs的增殖。與正常血清刺激人PASMCs比較，黃疸血清刺激人PASMCs的體外增殖能力明顯增強。在不同時間段內(nèi)，48h內(nèi)的增殖能力最強，雖然48h到72h增殖能力較弱，但仍比24h內(nèi)增殖能力強。說明了黃疸患者血清可以促進人PASMCs在體外增殖。 3.黃疸患者血清促進人PASMCs發(fā)生表型轉(zhuǎn)換作用24h后，western bolt檢測結果發(fā)現(xiàn)，與正常血清相比，黃疸血清刺激α-SM-actin蛋白變化不明顯，而calponin蛋白卻表達升高。但是，隨著作用時間延長，48h和72h后，黃疸血清刺激α-SM-actin和calpnion蛋白的表達均明顯減少。 4. luminex-XMAP液相芯片檢測結果顯示61種細胞因子中，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表達明顯升高，而其他的細胞因子（如IL-3、IL-7、TRAC、TRAIL、GRO、MCP-1、MCP-2、ENA-78及sCD40L）表達明顯下降。 結論： 1.改良的成人PASMCs培養(yǎng)方法克服了成人肺動脈平滑肌細胞培養(yǎng)困難的問題，同時將人PASMCs應用于實驗更符合人體形態(tài)學及生物學特征。 2.黃疸患者血清促進人PASMCs體外發(fā)生表型轉(zhuǎn)換和增殖。 3.通過luminex-XMAP液相芯片技術篩選出肝硬化患者血清中的變化較為明顯的細胞因子，為進一步研究肝源性肺疾病機制提供科學依據(jù)。
[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 偽 - 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 偽 - 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 偽 - SM - actin was not obvious , but calpain protein was increased . However , the expression of 偽 - 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 - 偽 , IL - 28A , LIF , I - 309 , IL - 15 , IL - 28A , LIF , I - 309 , MIP - 1偽 , IL - 28A , LIF , I - 309 , MIP - 1偽 , INF - 緯 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 .
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575

【參考文獻】

相關期刊論文 前3條

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3 Gokhan Tumgor;;Cirrhosis and hepatopulmonary syndrome[J];World Journal of Gastroenterology;2014年10期

本文編號：2089403