【摘要】：目的:建立PLVX-CD19-CD28-CD137-TCRl慢病毒載體并進(jìn)行鑒定,討論構(gòu)建的慢病毒載體轉(zhuǎn)染目的T細(xì)胞在急性B淋巴細(xì)胞白血病中的治療情況,進(jìn)一步確定CD19靶向抗原在B淋巴細(xì)胞白血病治療的重要作用,聯(lián)合CD28、CD137抗原共刺激T細(xì)胞,加強(qiáng)T細(xì)胞增殖能力,發(fā)揮更好的抗腫瘤效果。方法:通過(guò)基因工程和分子生物學(xué)實(shí)驗(yàn)技術(shù)構(gòu)建CD19-28-137-TCRl目標(biāo)基因序列,通過(guò)慢病毒載體與基因序列相結(jié)合。通過(guò)PCR擴(kuò)增技術(shù)、瓊脂糖凝膠電泳檢測(cè)CD19-28-137-TCRl的表達(dá)并對(duì)目標(biāo)基因序列進(jìn)行驗(yàn)證。將構(gòu)建好的基因載體用CaCL2法體外轉(zhuǎn)染到293FT細(xì)胞中,運(yùn)用免疫熒光的方法48 h時(shí)觀察目標(biāo)載體轉(zhuǎn)染效率和進(jìn)行慢病毒滴度的計(jì)算。通過(guò)Weston blot方法檢測(cè)酶切pLVX-CD19-28-137-TCRl在293FT細(xì)胞蛋白中的表達(dá)。在云南省第一人民醫(yī)院血液科尋找1名適合并愿意做CAR-T細(xì)胞治療的B-ALL患者,分離病人T淋巴細(xì)胞,用pLVX-CD19-28-137-TCRl慢病毒載體進(jìn)行體外轉(zhuǎn)染T細(xì)胞。將CD19-28-137-TCRl抗體基因?qū)隩細(xì)胞。擴(kuò)大培養(yǎng)建立好的CD19-CAR-T細(xì)胞。同時(shí),通過(guò)此法由北京免疫治療研究院構(gòu)建臨床治療所用CD19-CAR-T細(xì)胞,對(duì)患者進(jìn)行細(xì)胞回輸。治療后,流式細(xì)胞術(shù)對(duì)病人骨髓細(xì)胞流式免疫分型,骨髓活檢切片免疫組化分析,電鏡觀察骨髓細(xì)胞來(lái)判斷臨床效應(yīng)。結(jié)果:通過(guò)PCR擴(kuò)增的方法檢測(cè)到CD19-28-137-TCRl得到穩(wěn)定的表達(dá)。通過(guò)CaCl2法成功將慢病毒載體轉(zhuǎn)染到293FT細(xì)胞中并得到成功包裝。熒光顯微鏡觀察慢病毒對(duì)T細(xì)胞的轉(zhuǎn)染效率近90%,慢病毒滴度計(jì)算結(jié)果為5.5X107TU/ml。Weston blot方法檢測(cè)經(jīng)過(guò)酶切質(zhì)粒包裝后的293FT細(xì)胞蛋白以及沒(méi)有進(jìn)行酶切質(zhì)粒的293FT細(xì)胞蛋白CD19-CAR基因的表達(dá)。結(jié)果為β-actin蛋白條帶很清晰,經(jīng)過(guò)酶切后的質(zhì)粒蛋白得到在293FT細(xì)胞中得到相對(duì)較高的表達(dá),而沒(méi)有經(jīng)過(guò)酶切的CD19單鏈抗體基因表達(dá)不明顯。應(yīng)用CD19-CAR-T在1例急性B淋巴細(xì)胞白血病臨床治療結(jié)果中,電子顯微鏡觀察骨髓增生活躍,病人經(jīng)CAR-T治療后伴有骨髓纖維化產(chǎn)生。CAR-T治療后比治療前淋巴細(xì)胞亞群淋巴細(xì)胞、粒細(xì)胞、單核細(xì)胞、原始-髓系前體細(xì)胞下降,有核紅區(qū)域細(xì)胞、CD34+占有核細(xì)胞上升。免疫組化結(jié)果為,CD2、CD3、CD5、CD7、CD19、CD20抗體均為陰性不表達(dá),病變細(xì)胞表達(dá)TdT,部分表達(dá)Pax-5抗體以及Ki-67抗體,結(jié)果顯示為早期病變的B細(xì)胞。結(jié)論:成功構(gòu)建pLVX-CD19-28-137-TCRl慢病毒載體,并經(jīng)過(guò)瓊脂糖凝膠電泳進(jìn)行鑒定PLVX-CD19-28-137-TCRl慢病毒載體,WB成功檢測(cè)慢病毒用293FT細(xì)胞得到成功包裝,病毒滴度相對(duì)較高達(dá)到5.5X107TU/ml。pLVX-CD19-28-137-TCRl成功轉(zhuǎn)染目的T細(xì)胞,且對(duì)B淋巴細(xì)胞白血病的治療起到一定的作用,但副作用伴隨有骨髓纖維化。
[Abstract]:Objective: to establish and identify the PLVX-CD19-CD28-CD137-TCRl lentivirus vector and to discuss the therapeutic effect of the constructed lentivirus vector transfection target T cells in acute B lymphocytic leukemia. To further determine the important role of CD19 target antigen in the treatment of B lymphocyte leukemia, combined with CD28,CD137 antigen to stimulate T cells, enhance the ability of T cell proliferation, play a better anti-tumor effect. Methods: the target gene sequence of CD19-28-137-TCRl was constructed by gene engineering and molecular biology experiment, and the gene sequence was combined with lentivirus vector. The expression of CD19-28-137-TCRl was detected by agarose gel electrophoresis and the target gene sequence was verified by PCR amplification. The constructed gene vector was transfected into 293FT cells by CaCL2 method in vitro. The transfection efficiency of the target vector and the calculation of the lentivirus titer were observed by immunofluorescence method at 48 h. The expression of pLVX-CD19-28-137-TCRl in 293FT cells was detected by Weston blot method. A suitable B-ALL patient who was willing to be treated with CAR-T cells was selected in hematology department of the first people's Hospital of Yunnan Province. The T lymphocytes were isolated and transfected with pLVX-CD19-28-137-TCRl lentivirus vector in vitro. CD19-28-137-TCRl antibody gene was introduced into T cells. CD19-CAR-T cells were established by expanded culture. At the same time, the CD19-CAR-T cells for clinical therapy were constructed by this method. After treatment, flow cytometry was used to determine the clinical effects of bone marrow cells by flow cytometry, immunohistochemical analysis of bone marrow biopsy sections and observation of bone marrow cells by electron microscope. Results: stable expression of CD19-28-137-TCRl was detected by PCR amplification. The lentivirus vector was successfully transfected into 293FT cells by CaCl2 and packaged successfully. Fluorescence microscope was used to observe the transfection efficiency of lentivirus on T cells. The results of lentivirus titer calculation were 5.5X107TU/ml.Weston blot method to detect the expression of 293FT cell protein and 293FT cell protein CD19-CAR gene which were packaged by enzyme digestion plasmid and 293FT cell protein without plasmids digested by lentivirus. The results showed that the band of 尾-actin protein was clear, the plasmid protein digested by enzyme was relatively high expression in 293FT cells, but the expression of CD19 single chain antibody gene without enzyme digestion was not obvious. Bone marrow hyperplasia was observed by electron microscopy in a case of acute B lymphocyte leukemia treated with CD19-CAR-T. After CAR-T treatment, the lymphocyte subsets, granulocytes, monocytes, primordial progenitor cells, nucleated red region cells decreased after CAR-T treatment. CD34 has increased in nuclear cells. The results of immunohistochemistry showed that the CD2,CD3,CD5,CD7,CD19,CD20 antibody was negative, and the TdT, partial expression of Pax-5 antibody and the Ki-67 antibody were expressed in the pathological cells. The results showed that the cells were B cells with early pathological changes. Conclusion: pLVX-CD19-28-137-TCRl lentivirus vector was successfully constructed and PLVX-CD19-28-137-TCRl lentivirus vector was identified by agarose gel electrophoresis. WB was successfully packaged with 293FT cells. The titer of the virus was relatively high and the target T cells were successfully transfected with 5.5X107TU/ml.pLVX-CD19-28-137-TCRl, which played a certain role in the treatment of B lymphocytic leukemia, but the side effects were accompanied by bone marrow fibrosis.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.7

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本文編號(hào)：2311596