本文選題：人臍靜脈內(nèi)皮細胞 + 淋巴管內(nèi)皮表型��； 參考：《山東大學》2012年碩士論文

【摘要】：研究背景：淋巴管是心血管循環(huán)系統(tǒng)的重要輔助系統(tǒng),在調(diào)節(jié)體內(nèi)滲透壓、維持內(nèi)環(huán)境穩(wěn)定以及免疫、炎癥反應中發(fā)揮重要作用。淋巴管新生(lymphangiogenesis)與機體組織許多病理生理過程密切相關,尤其是在炎癥反應、創(chuàng)傷修復以及腫瘤轉移等方面發(fā)揮重要作用。因此,探尋淋巴管新生機制對于上述疾病的治療具有重要意義。 以往的研究認為,淋巴管新生的途徑主要有兩個方面：原有的淋巴管在血管內(nèi)皮生長因子C (VEGF-C)的作用下以出芽方式形成新的淋巴管；外周血內(nèi)皮祖細胞遷移到局部組織,在VEGF-C等的誘導下轉分化為淋巴管內(nèi)皮細胞(lymphatic endothelial cells, LECs)。近期的研究發(fā)現(xiàn),許多炎性介質與淋巴管標志物(Prox-1、Podoplanin、VEGFR-3、LYVE-1等)的表達和淋巴管新生有密切聯(lián)系,例如：LPS、TNF-α、IFN-γ、IL-1、IL-3、IL-6等。然而,炎性介質調(diào)節(jié)淋巴管內(nèi)皮表型的作用機制尚不十分清楚。 文獻報道,炎性介質LPS、IL-3等能刺激內(nèi)皮細胞的NF-κB的P50同源二聚體途徑誘導內(nèi)皮細胞淋巴管標志物的表達；另有文獻認為,血管內(nèi)皮細胞(blood endothelial cells, BECs)在炎性介質IFN-γ、TNF-α的作用下表達IL-3,間接通過IL-3刺激淋巴管標志物的表達。IL-1是許多病理過程中重要的炎性因子,能否通過NF-κB途徑誘導內(nèi)皮細胞表達淋巴管表型即向淋巴管內(nèi)皮細胞轉分化?尚未見報道。本文重點研究了IL-1β、IL-3誘導血管內(nèi)皮細胞轉分化為淋巴管內(nèi)皮細胞的調(diào)節(jié)作用。 目的：本實驗采用炎性介質IL-1β和IL-3分別處理分離培養(yǎng)的人臍靜脈內(nèi)皮細胞(human umbilical vein endothelial cells, HUVECs)和人臍靜脈內(nèi)皮細胞株CRL-1730,研究誘導或維持內(nèi)皮細胞的淋巴管內(nèi)皮表型特性的關鍵因素。 方法：①取臍帶,按常規(guī)分離培養(yǎng)原代HUVECs,細胞融合后觀察并記錄其形態(tài),用vWF相關抗原的免疫細胞化學染色驗證是否為BECs,傳代進行后續(xù)實驗；取人臍靜脈內(nèi)皮細胞株CRL-1730,按同樣條件培養(yǎng)。②采用RT-PCR和免疫細胞化學法檢測HUVECs和CRL-1730細胞株對淋巴管標志物Prox-1、VEGFR-3、 Podoplanin、LYVE-1的表達情況。③分別用IL-1p或IL-3刺激CRL-1730細胞株一定時間,觀察刺激前后細胞形態(tài)的改變；采用RT-PCR和免疫細胞化學法檢測細胞對淋巴管標志物的表達情況。④采用Real-time PCR法檢測CRL-1730細胞株經(jīng)IL-1β刺激前、后對淋巴管標志物mRNA表達量的差異。⑤將NF-κB阻斷劑PDTC加入到培養(yǎng)的CRL-1730細胞株中,1h后再加入IL-1β刺激一定時間后,采用免疫細胞化學法檢測細胞NF-κB活性改變,并用RT-PCR法檢測CRL-1730細胞株對淋巴管內(nèi)皮標志物的表達情況。 結果：①RT-PCR和免疫細胞化學法發(fā)現(xiàn)：分離培養(yǎng)的HUVECs表達淋巴管標志物,而CRL-1730細胞株則不表達淋巴管標志物。②CRL-1730細胞株經(jīng)IL-1β或IL-3刺激24h后,生長匯合的內(nèi)皮細胞形態(tài)由鵝卵石狀變?yōu)殚L梭形；RT-PCR和免疫細胞化學法證實表達淋巴管標志物。③Real-time PCR檢測結果顯示：與未經(jīng)刺激的分離培養(yǎng)的HUVECs相比,未經(jīng)刺激的內(nèi)皮細胞株CRL-1730對淋巴管內(nèi)皮標志物Prox-1、Podoplanin、VEGFR-3、LYVE-1的mRNA表達量極少；經(jīng)IL-1β刺激1.5h后表達量顯著升高(表1,圖10),P0.05。進一步驗證了之前刺激實驗做出的RT-PCR結果。④CRL-1730細胞株在加入NF-κB阻斷劑PDTC之后再用炎性因子IL-1β刺激,免疫細胞化學和RT-PCR法檢測其呈陰性表達。 結論：IL-1p和IL-3均能誘導血管內(nèi)皮細胞表達淋巴管內(nèi)皮表型；IL-1p能通過NF-κB途徑誘導BECs表達淋巴管內(nèi)皮表型。 意義：研究炎性介質誘導血管內(nèi)皮轉分化為淋巴管內(nèi)皮細胞作用,對于解釋炎癥環(huán)境下淋巴管形成機制具有重要意義,并為治療淋巴管生成或生成障礙相關疾病奠定基礎。
[Abstract]:Background : lymphatic vessel is an important auxiliary system of cardiovascular circulatory system , plays an important role in regulating osmotic pressure in vivo , maintaining homeostasis of internal environment and immune and inflammatory response .

Previous studies suggest that there are two main pathways for lymphatic angiogenesis : the original lymphatic vessels form new lymphatic vessels in budding manner under the action of vascular endothelial growth factor C ( VEGF - C ) ;
Peripheral vascular endothelial progenitor cells migrate to local tissues and differentiate into lymphatic endothelial cells ( LECs ) under the induction of VEGF - C and the like . Recent studies have found that many inflammatory mediators are closely related to the expression of lymphatic markers ( Prox - 1 , Podopathy , VEGFR - 3 , LYVE - 1 , etc . ) , such as LPS , TNF - 偽 , IFN - 緯 , IL - 1 , IL - 3 , IL - 6 , etc . However , the mechanism of inflammatory mediators for modulating lymphatic endothelial phenotype is not very clear .

It is reported that inflammatory mediators LPS , IL - 3 and the like can stimulate the expression of endothelial cell lymphoma markers by stimulating the P50 homologous dimer pathway of NF - 魏B in endothelial cells ;
IL - 1 is an important inflammatory factor in many pathological processes . It has not been reported that IL - 1 is an important inflammatory factor in many pathological processes . It has not been reported that IL - 1尾 and IL - 3 can induce endothelial cells to differentiate into lymphatic endothelial cells .

Objective : To investigate the key factors in the induction or maintenance of endothelial cell endothelial phenotype in cultured human umbilical vein endothelial cells ( HUVEC ) and human umbilical vein endothelial cells ( CRL - 1730 ) using inflammatory mediators IL - 1尾 and IL - 3 respectively .

Methods : ( 1 ) The umbilical cord was isolated and cultured in vitro . The morphology was observed and recorded after cell fusion .
The expression of Prox - 1 , VEGFR - 3 , Podopathy and LYVE - 1 was detected by RT - PCR and immunohistochemical method .
The expression of lymphocyte markers was detected by RT - PCR and immune cytochemical method . The mRNA expression of lymphocytes was detected by Real - time PCR .

Results : 鈶，

本文編號：2054403