本文選題：免疫磁珠分離　切入點：B細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目前在全球范圍內(nèi)，感染乙型肝炎病毒的人大約有20億，我國約有9300萬人。乙型肝炎病毒嚴(yán)重威脅著人類的健康。慢性乙型肝炎的治療目前仍然缺乏行之有效的辦法和藥物。HBV病毒本身不直接破壞肝臟細(xì)胞，而肝細(xì)胞的損傷主要是由于機(jī)體為了清除感染了HBV的肝細(xì)胞的免疫反應(yīng)導(dǎo)致【1】。因此，從免疫學(xué)的角度對慢性乙型肝炎的研究仍然具有重要意義。 目的 以細(xì)胞膜受體CXCR5為共同抗原標(biāo)志，應(yīng)用免疫磁珠分選法建立Tfh細(xì)胞和B細(xì)胞共培養(yǎng)體系，在成功構(gòu)建共培養(yǎng)體系的基礎(chǔ)上體外觀察培養(yǎng)體系與人肝癌細(xì)胞系HepG2.2.15細(xì)胞混合培養(yǎng)前后共刺激因子的變化。為進(jìn)一步體外研究細(xì)胞之間的相互關(guān)系奠定基礎(chǔ)。 方法 1.研究對象：慢性乙型肝炎肝硬化行脾切除術(shù)患者15例（男8，女7）； 2.流式細(xì)胞儀檢測：獲取慢性乙型肝炎肝硬化行脾切除術(shù)患者術(shù)前外周血20ml，術(shù)中取脾臟組織10g左右，分離獲得淋巴細(xì)胞，以相應(yīng)的抗體標(biāo)記，以流式細(xì)胞儀檢測外周血來源與脾臟來源的Tfh細(xì)胞表型； 3.以免疫磁珠方法構(gòu)建Tfh細(xì)胞和B細(xì)胞共培養(yǎng)體系：采用梯度離心法分離15例慢性乙肝病毒感染者脾臟單個核細(xì)胞(PBMC)，以細(xì)胞膜受體CXCR5為抗原標(biāo)志，采用免疫磁珠分選法分離CXCR5+細(xì)胞，通過流式細(xì)胞儀檢測CXCR5+細(xì)胞純度和組成； 4.混合培養(yǎng)：將CXCR5+細(xì)胞和人肝癌細(xì)胞系HepG2.2.15細(xì)胞在體外混合培養(yǎng)1周； 5.流式細(xì)胞儀檢測：CXCR5+細(xì)胞和人肝癌細(xì)胞系HepG2.2.15細(xì)胞在體外混合培養(yǎng)1周后，應(yīng)用流式細(xì)胞儀檢測Tfh細(xì)胞表型變化； 6.混合培養(yǎng)液上清檢測：混合培養(yǎng)1周后，應(yīng)用ELISA法檢測培養(yǎng)上清液HBeAb； 7.混合培養(yǎng)液上清HBV DNA水平測定：將收集的上清液經(jīng)DNA提取液處理后進(jìn)行PCR擴(kuò)增，按照HBV DNA熒光定量PCR檢測試劑盒操作步驟進(jìn)行。 8.混合培養(yǎng)細(xì)胞CXCR5mRNA水平測定：（1）提取總RNA：按照高純總RNA快速提取試劑盒說明書進(jìn)行。（2）合成cDNA：以每10ul反應(yīng)體系400ng總RNA按逆轉(zhuǎn)錄試劑盒說明書進(jìn)行合成。（3） PCR擴(kuò)增： CXCR5引物： F:5-CTCCTCTCCATCCACATCACCT-3，R:5-CGTTTCTGCTTGGTTCTCTTGG-3。反應(yīng)條件為：95℃30S；95℃5S，60℃31S，40個循環(huán)。2-△△CT方法分析基因表達(dá)水平，B-actin為內(nèi)參。 9.統(tǒng)計學(xué)方法：用SPSS17.0軟件進(jìn)行分析，采用t檢驗和方差分析，以P0.05為差異有統(tǒng)計學(xué)意義。 結(jié)果 1.同一患者外周血來源的Tfh細(xì)胞和脾臟來源的Tfh細(xì)胞表型存在差異。外周血來源的Tfh細(xì)胞ICOS，PDL1，PDL1表達(dá)低于脾臟Tfh細(xì)胞,差異具有統(tǒng)計學(xué)意義（P0.05），而CCR7表達(dá)高于脾臟Tfh細(xì)胞，差異具有統(tǒng)計學(xué)意義（P0.05）； 2.同一患者外周血來源的Tfh細(xì)胞分泌的細(xì)胞因子IFN-γ（8.32±1.21）明顯高于脾臟來源的Tfh細(xì)胞分泌IFN-γ（3.83±0.32），差異具有統(tǒng)計學(xué)意義（P0.05）； 3.應(yīng)用磁珠分選法能夠成功獲得CXCR5+細(xì)胞：用流式細(xì)胞儀鑒定細(xì)胞的純度和構(gòu)成：CXCR5+細(xì)胞純度為85.53±5.77%，其中tfh細(xì)胞占:23.8±7.4%,B細(xì)胞占:35.6±7.6%; 4.CXCR5+細(xì)胞和人肝癌細(xì)胞系HepG2.2.15細(xì)胞在體外混合培養(yǎng)1周后，應(yīng)用流式細(xì)胞儀檢測Tfh細(xì)胞表型的變化，發(fā)現(xiàn)PD1, PDL1，ICOS均明顯低于培養(yǎng)前，差異具有統(tǒng)計學(xué)意義（P0.05）。Tfh細(xì)胞分泌因子IFN-γ的表達(dá)情況也明顯高于培養(yǎng)前，差異具有統(tǒng)計學(xué)意義CCR7在培養(yǎng)后明顯高于培養(yǎng)前和外周血來源的Tfh細(xì)胞的表達(dá)量，，差異具有統(tǒng)計學(xué)意義（P0.05）； 5.混合培養(yǎng)1周后上清液HBeAb定量檢測結(jié)果，實驗組HBeAb(0.768±0.094)均高于空白對照組HBeAb(0.478±0.088),差異具有統(tǒng)計學(xué)意義； 6.混合培養(yǎng)液上清HBV-DNA定量檢測結(jié)果:實驗組HBV-DNA檢測結(jié)果明顯低于空白對照組，HBV-DNA抑制率為33%； 7.將CXCR5+細(xì)胞和人肝癌細(xì)胞系HepG2.2.15細(xì)胞在體外混合培養(yǎng)1周后，檢測CXCR5+細(xì)胞表面CXCR5mRNA的表達(dá)情況，實驗組和空白對照組相比無顯著差異。 結(jié)論 1.脾臟來源的Tfh細(xì)胞和外周血來源的Tfh細(xì)胞共刺激分子表達(dá)存在差異，CXCR5+細(xì)胞和人肝癌細(xì)胞系HepG2.2.15細(xì)胞混合培養(yǎng)后發(fā)現(xiàn)脾臟來源的Tfh細(xì)胞細(xì)胞表型有向外周血來源的Tfh細(xì)胞表型轉(zhuǎn)變的趨勢； 2.使用間接免疫磁珠分離法可成功建立CXCR5+B淋巴細(xì)胞和Tfh細(xì)胞的共培養(yǎng)體系，為進(jìn)一步體外研究細(xì)胞之間相互關(guān)系奠定基礎(chǔ)。 3.CXCR5+細(xì)胞對HBV-DNA的復(fù)制具有抑制作用，HBV-DNA抑制率為33%，對HBsAg，HBeAg也具有抑制作用，通過擴(kuò)增CXCR5后發(fā)現(xiàn)實驗組和對照組無明顯差異，說明CXCR5+細(xì)胞對HBV-DNA復(fù)制的影響不是通過高表達(dá)CXCR5的途徑來實現(xiàn)的，具體的作用機(jī)制有待進(jìn)一步的研究。
[Abstract]:At present , there are about 2 billion people infected with hepatitis B virus worldwide . There are about 93 million people in our country . Hepatitis B virus is a serious threat to human health . The treatment of chronic hepatitis B is still lack of effective methods and medicines . HBV virus itself does not directly destroy liver cells , and the damage of liver cells is caused by the immune response of liver cells infected with HBV . Therefore , the study of chronic hepatitis B is still of great significance from the perspective of immunology .

Purpose

Based on the successful construction of the co - culture system , the changes of costimulatory factors were observed in the culture system and the human hepatoma cell line HepG2.2 . 15 .

method

1 . Subjects : 15 patients with chronic hepatitis B cirrhosis ( male 8 , female 7 ) ;


2 . Flow cytometry was used to detect the peripheral blood flow in 20 ml of peripheral blood before operation in patients with chronic hepatitis B cirrhosis .


3 . Using immune magnetic bead method to construct the system of the co - culture of T cell and B cells : 15 cases of chronic hepatitis B virus infected with spleen mononuclear cells ( PBMC ) were separated by gradient centrifugation , the cell membrane receptor ' s expression was used as the antigen marker , the cells were separated by immunomagnetic bead sorting method , and the purity and composition were detected by flow cytometry .


4 . mixing culture : carrying out mixing culture for 1 week ;


5 . Flow cytometry was used to detect the phenotype changes of the cell line HepG5 + cells and human hepatoma cell line HepG2.2 . 15 after 1 week in vitro .


6 . supernatant of mixed culture solution : after 1 week of mixed culture , ELISA method was used to test the culture supernatant ;


7 . measuring HBV DNA level on the mixed culture medium : carrying out PCR amplification after the collected supernatant is processed by a DNA extraction solution , and carrying out the operation steps of the kit according to the HBV DNA fluorescence quantitative PCR detection kit .

8 . Assay : ( 1 ) extracting total RNA : ( 1 ) extracting total RNA : ( 1 ) extracting total RNA : ( 2 ) synthesizing cDNA : synthesizing the total RNA of 400ng total RNA in every 10 ul reaction system according to the instructions of reverse transcription kit .
The gene expression level was analyzed by 2 - 鈻

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