本文選題：庫(kù)普弗細(xì)胞 + 腺病毒載體�。� 參考：《蘇州大學(xué)》2007年碩士論文

【摘要】： 目的:(1)分離純化并鑒定SD大鼠庫(kù)普弗細(xì)胞(Kupffer Cell,KC),得到較高純度及活力的KC。(2)重組腺病毒Adv-IL-12體外轉(zhuǎn)染KC,檢測(cè)IL-12及MHC-Ⅰ類分子(H2-Kb)、MHC-Ⅱ類分子(I-Ab)的表達(dá)情況。(3)觀察Adv-IL-12轉(zhuǎn)染的KC對(duì)脾T細(xì)胞免疫功能的影響,為靶向治療腫瘤肝內(nèi)微轉(zhuǎn)移的實(shí)驗(yàn)研究提供依據(jù)。 方法: (1)采用Ⅳ型膠原酶、鏈霉蛋白酶E (PronaseE)灌注消化,Nycodenz密度梯度離心法得到SD大鼠肝臟非實(shí)質(zhì)細(xì)胞(nonparenchymal cells,NPC),再以選擇性貼壁法純化KC。以臺(tái)盼藍(lán)拒染實(shí)驗(yàn)判斷細(xì)胞產(chǎn)率和存活率;以細(xì)胞自發(fā)熒光現(xiàn)象、吞噬實(shí)驗(yàn)及ED-2單克隆抗體染色法對(duì)KC進(jìn)行鑒定。(2)24孔細(xì)胞培養(yǎng)板中培養(yǎng)KC,細(xì)胞密度為1×106/ml,共設(shè)置3組,分別為KC細(xì)胞對(duì)照組(下稱細(xì)胞對(duì)照組)、KC+Adv-EGFP病毒對(duì)照組(下稱病毒對(duì)照組)和KC+Adv-IL-12試驗(yàn)組(下稱實(shí)驗(yàn)組),每組設(shè)三個(gè)復(fù)孔。分別于轉(zhuǎn)染后24h、48h收取3組細(xì)胞上清,以ELISA試劑盒檢測(cè)細(xì)胞上清中IL-12的表達(dá)量;轉(zhuǎn)染后48h收集各組細(xì)胞,以半定量RT-PCR法檢測(cè)MHCⅠ類分子(H-2Kb)及MHCⅡ類分子(I-Ab)mRNA對(duì)應(yīng)于內(nèi)參照三磷酸甘油醛(GAPDH)的相對(duì)含量。(3)常規(guī)制備大鼠脾淋巴細(xì)胞(Splenic lymphocyte,SLC),調(diào)整密度為2×106/ml,加入ConA(終濃度為5μg/ml)刺激培養(yǎng)72小時(shí)后,與事先以重組IL-12腺病毒轉(zhuǎn)染的KC混合培養(yǎng),SLC與KC之比為10:1,分4組:SLC+Adv-IL-12-KC組(A組)、SLC+Adv-EGFP-KC組(B組)、SLC+KC組(C組)、單獨(dú)SLC組(D組),每組設(shè)三個(gè)復(fù)孔。共同培養(yǎng)72h后,收集各孔培養(yǎng)上清及SLC,ELISA法測(cè)定培養(yǎng)上清中IFN-γ和IL-4蛋白的水平;以熒光標(biāo)記的抗大鼠CD4、CD8單抗染色,流式細(xì)胞儀測(cè)定各組SLC中CD4T、CD8T細(xì)胞亞群變化。 結(jié)果: (1)實(shí)驗(yàn)中每只SD大鼠可得到3.5-5.0×107個(gè)KC,細(xì)胞的存活率可達(dá)到93%以上;ED-2單抗染色鑒定,KC純度達(dá)95%以上。(2)50 MOI劑量的重組腺病毒感染KC, ELISA檢測(cè):轉(zhuǎn)染后24h,實(shí)驗(yàn)組上清IL-12濃度即明顯升高,達(dá)對(duì)照組的10倍左右,48h測(cè)定IL-12濃度繼續(xù)升高,實(shí)驗(yàn)組IL-12濃度可達(dá)1000pg/ml以上水平,與對(duì)照組比較差別具有顯著統(tǒng)計(jì)學(xué)意義;而細(xì)胞對(duì)照組和病毒對(duì)照組KC也有一定的基礎(chǔ)分泌,前者IL-12分泌量略低于后者,但差別不具有統(tǒng)計(jì)學(xué)意義。半定量RT-PCR法檢測(cè)表明實(shí)驗(yàn)組H-2Kb /GAPDH和I-Ab /GAPDH比值分別為0.835,0.739,分別上調(diào)了1.5倍(H-2Kb)和2.0倍(I-Ab),與病毒對(duì)照組和細(xì)胞對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義,而病毒對(duì)照組和細(xì)胞對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)意義。(3)ELISA檢測(cè)可見: A組、B組、C組和D組培養(yǎng)上清中IFN-γ含量依次為2026.29±103.37、988.55±94.39、1005.36±120.18、505.12±55.28(pg/mL),而IL-4含量依次為206.94±22.43、320.57±34.73、349.84±49.61、828.26±62.83(pg/mL)。FCM檢測(cè)顯示:A組SLC中CD4T細(xì)胞比例、T細(xì)胞總數(shù)(CD4T細(xì)胞和CD8T細(xì)胞之和)及CD4T/ CD8T比值均顯著增大,與其他3組相比有顯著差異,但CD8T細(xì)胞比例在各組間無(wú)明顯差異;B組與C組、D組之間在各項(xiàng)指標(biāo)上的差異不具有統(tǒng)計(jì)學(xué)意義;C組的CD4T細(xì)胞比例及T細(xì)胞總數(shù)高于D組,但兩組CD8T細(xì)胞比例及CD4T/CD8T比值之間無(wú)明顯差別。 結(jié)論: (1)采用Ⅳ型膠原酶、PronaseE灌注消化,Nycodenz密度梯度離心及選擇性貼壁法能夠分離出較高純度和活力的KC,能滿足進(jìn)一步的實(shí)驗(yàn)要求。(2)在體外腺病毒載體能有效的轉(zhuǎn)染KC;IL-12基因修飾后,KC能高表達(dá)IL-12蛋白;其MHCⅠ和MHCⅡ分子基因轉(zhuǎn)錄增強(qiáng),說(shuō)明IL-12基因修飾能夠上調(diào)KC的抗原提呈能力。(3)IL-12基因修飾的KC細(xì)胞與脾淋巴細(xì)胞混合培養(yǎng),能顯著增加混合淋巴細(xì)胞中CD4T細(xì)胞的比例,而對(duì)CD8T細(xì)胞無(wú)明顯影響,結(jié)果導(dǎo)致CD4T/ CD8T比值增大;由IL-12誘導(dǎo)的CD4T細(xì)胞增殖主要分化為Th1細(xì)胞,培養(yǎng)上清中Th1型細(xì)胞因子IFN-γ的含量顯著增加反映了這一變化,與此同時(shí)Th2方向的分化受到抑制,并表現(xiàn)為Th2型細(xì)胞因子IL-4分泌量的顯著減少。
[Abstract]:Objective: (1) to isolate and purify and identify Kupffer Cell (KC) in SD rats, and to obtain KC. (2) recombinant adenovirus Adv-IL-12 with high purity and vigor to transfect KC in vitro, to detect the expression of IL-12 and MHC- class I molecules (H2-Kb), MHC- class II molecules (I-Ab). (3) observe the effect of transfection on the immune function of spleen cells. To provide evidence for the experimental study of tumor micrometastasis in liver.
Methods: (1) using type IV collagenase, streptomycin E (PronaseE) perfusion and digestion, Nycodenz density gradient centrifugation was used to obtain SD rat liver non parenchymal cells (nonparenchymal cells, NPC), and then purified KC. with trypan blue exclusion test to determine the cell yield and survival rate by selective adherence method, and the cell spontaneous fluorescence phenomenon, phagocytic experiment and ED-2. KC was identified by monoclonal antibody staining. (2) 24 hole cell culture plates were cultured for KC, and the cell density was 1 x 106/ml. A total of 3 groups were set up, respectively, the control group of KC cells (the lower cell control group), the KC+Adv-EGFP virus control group (the lower virus control group) and the KC+Adv-IL-12 test group (hereinafter referred to as the experimental group) with three compound holes in each group. 24 after transfection, respectively. H, 48h collected 3 groups of cell supernatants and detected the expression of IL-12 in the cell supernatant by ELISA kit. After transfection, 48h collected all groups of cells and detected the MHC class I molecules (H-2Kb) and MHC class II molecules (I-Ab) mRNA corresponding to the relative content of glyceraldehyde (GAPDH) of internal reference three phosphoric acid (GAPDH) by semi quantitative RT-PCR method. (3) normal rat spleen lymphocytes were prepared. Phocyte, SLC), the adjustment density was 2 x 106/ml, and the ConA (final concentration of 5 mu g/ml) was stimulated for 72 hours. The ratio of SLC to KC was 10:1, and the ratio of SLC to KC was 10:1, which was divided into 4 groups: SLC+Adv-IL-12-KC group (A group), individual group (Group), each group had three compound holes. Co culture After 72h, the level of IFN- gamma and IL-4 protein in culture supernatant was measured by the culture supernatant of each hole, and the level of IFN- gamma and IL-4 protein in the culture supernatant. The CD4T and CD8T cell subgroups of SLC in each group were measured by the fluorescent labeled anti CD4, CD8 mAb staining and the flow cytometry.
Results: (1) each SD rat could get 3.5-5.0 x 107 KC, the survival rate of the cell could reach more than 93%, and the purity of KC was above 95%. (2) the recombinant adenovirus infected with the 50 MOI dose KC, ELISA detection: 24h after transfection, the IL-12 concentration of the experimental group Rose obviously, reaching about 10 times of the control group and 48h determination IL-12 concentration. The concentration of IL-12 in the experimental group was up to 1000pg/ml, and the difference between the control group and the control group had significant statistical significance, while the cell control group and the virus control group had a certain basal secretion, the former IL-12 secretion was slightly lower than the latter, but the difference was not statistically significant. The semi quantitative RT-PCR test showed that the experimental group was H-2Kb /GAPD. The ratio of H and I-Ab /GAPDH was 0.835,0.739, respectively up 1.5 times (H-2Kb) and 2 times (I-Ab), compared with the virus control group and cell control group, but there was no significant difference between the virus control group and the cell control group. (3) ELISA detection could be seen in A group, B group, C group and D group. 2026.29 + 103.37988.55 + 94.391005.36 + 120.18505.12 + 55.28 (pg/mL), while the content of IL-4 was 206.94 + 22.43320.57 + 49.61828.26 + 62.83 (pg/mL).FCM. There was no significant difference in the proportion of CD8T cells in each group, but there was no statistical difference between group B and group C and group D in each index. The proportion of CD4T cells and the total number of T cells in group C were higher than that in D group, but there was no significant difference between the proportion of CD8T cells and the ratio of CD4T/CD8T in the two groups.
Conclusion: (1) the use of type IV collagenase, PronaseE perfusion and digestion, Nycodenz density gradient centrifugation and selective adherence can separate the higher purity and vitality of KC, and can meet further experimental requirements. (2) KC can be transfected effectively by adenovirus vector in vitro, and KC can express high expression of IL-12 protein after IL-12 gene modification; and its MHC I and MHC II molecular basis The IL-12 gene modification could increase the antigen presentation ability of KC. (3) the mixed culture of KC cells modified by IL-12 gene and spleen lymphocyte can significantly increase the proportion of CD4T cells in mixed lymphocytes, but have no obvious effect on CD8T cells, resulting in the increase of CD4T/ CD8T ratio, and the proliferation of CD4T cells induced by IL-12 is the main factor. A significant increase in the content of Th1 type cytokine IFN- gamma in the culture supernatant reflects this change, at the same time, the differentiation of Th2 direction is inhibited, and the secretion of Th2 type cytokine IL-4 is significantly reduced.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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