【摘要】： 目的：初步研究RetroNectin對(duì)CIK細(xì)胞增值、表性變化及殺傷活性的影響，并探討其可能機(jī)制。 方法：采集人外周血單個(gè)核細(xì)胞，標(biāo)本分為兩組，對(duì)照組加入IFN-γ、IL-2、IL-1α、CD3mAb等細(xì)胞因子誘導(dǎo)其增殖，實(shí)驗(yàn)組另外加入RetroNectin誘導(dǎo)，倒置顯微鏡下觀察培養(yǎng)過程中細(xì)胞形態(tài)變化，采用活細(xì)胞計(jì)數(shù)法觀察CIK細(xì)胞增殖，分別繪制細(xì)胞增值曲線；流式細(xì)胞術(shù)檢測(cè)CIK細(xì)胞培養(yǎng)過程中的表型變化；LDH法檢測(cè)CIK細(xì)胞對(duì)腫瘤細(xì)胞K562細(xì)胞和PC-3細(xì)胞的殺傷活性；采用AnnexinV／PI染色，，流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況；流式細(xì)胞技術(shù)檢測(cè)不同培養(yǎng)條件下第4d、7d、11d及14d的CIK細(xì)胞周期。 結(jié)果：PBMC在體外經(jīng)過多種細(xì)胞因子的誘導(dǎo)后，能大量擴(kuò)增生成CIK細(xì)胞，培養(yǎng)14天，擴(kuò)增倍數(shù)可達(dá)96.13±5.03倍；CIK細(xì)胞中的CD3~+CD8~+、CD3~+CD56~+細(xì)胞比例隨培養(yǎng)時(shí)間的延長(zhǎng)較PBMC明顯增多(p＜0.05)；細(xì)胞的百分率則逐漸下降；CIK細(xì)胞對(duì)K562細(xì)胞和PC-3細(xì)胞均有較強(qiáng)的殺傷活性，兩者相比較，差異無統(tǒng)計(jì)學(xué)意義(p＞0.05)，并且隨著誘導(dǎo)時(shí)間的延長(zhǎng)，這種殺傷活性增強(qiáng)，在誘導(dǎo)培養(yǎng)的第14d殺傷活性最強(qiáng)。經(jīng)RetroNectin刺激14天后的RN-CIK細(xì)胞擴(kuò)增倍數(shù)可達(dá)330.46±7.96倍，培養(yǎng)的第7天起明顯高于普通培養(yǎng)方法(p＜0.05)；自培養(yǎng)的第7天，RN-CIK細(xì)胞中的CD25~+細(xì)胞比例較普通培養(yǎng)的CIK細(xì)胞明顯增多(p＜0.05)。但兩種培養(yǎng)方法對(duì)CIK細(xì)胞的CD3~+CD8~+、CD3~+CD56~+、CD3~+CD4~+表型及細(xì)胞殺傷活性的影響無明顯差異(p＞0.05)。在培養(yǎng)的第11d，RN-CIK細(xì)胞與普通培養(yǎng)的CIK細(xì)胞凋亡率無明顯差異(p＞0.05)。S期細(xì)胞的比例隨所培養(yǎng)時(shí)間的延長(zhǎng)逐漸升高，在培養(yǎng)的第7d達(dá)到最高，并且RN-CIK細(xì)胞中S期的細(xì)胞比例明顯高于普通培養(yǎng)的CIK細(xì)胞組(p＜0.05)。 結(jié)論：(1)體外應(yīng)用抗人CD3單抗、人重組IL-1α、人重組IFN-γ和人重組IL-2能誘導(dǎo)PBMC生成CIK細(xì)胞，CIK細(xì)胞對(duì)K562細(xì)胞和PC-3細(xì)胞均有較強(qiáng)的殺傷活性。(2)RetroNectin可以明顯增加CIK細(xì)胞的擴(kuò)增倍數(shù)，但是對(duì)CIK細(xì)胞的表型變化及殺傷活性影響不大。(3)RetroNectin能促進(jìn)細(xì)胞增殖的可能機(jī)制：促進(jìn)細(xì)胞之間的黏附，增強(qiáng)信號(hào)傳導(dǎo)；誘導(dǎo)T細(xì)胞活化；抵抗活化細(xì)胞凋亡；促使細(xì)胞由G1期進(jìn)入S期。
[Abstract]:Aim: to study the effect of RetroNectin on the proliferation, epigenetic change and cytotoxicity of CIK cells and its possible mechanism. Methods: human peripheral blood mononuclear cells (PBMC) were collected and divided into two groups. The control group was induced by IFN- 緯 -IL-2IL-2IL-1 偽 CD3mAb, and the experimental group was induced by RetroNectin. The morphological changes of the cells were observed under inverted microscope. The proliferation of CIK cells was observed by living cell count and the proliferation curve was plotted respectively. The phenotypic changes of CIK cells were detected by flow cytometry. The cytotoxicity of CIK cells to K562 cells and PC-3 cells was detected by flow cytometry. AnnexinV/PI staining and flow cytometry were used to detect the apoptosis of the cells, and the CIK cell cycle at the 4th day after 7 days and 14 days under different culture conditions was detected by flow cytometry. Results CIK cells were induced by many cytokines in vitro. After 14 days of culture, the percentage of CD3 ~ CD8 ~ + CD3 ~ CD56 ~ cells in CIK cells was 96.13 鹵5.03 times higher than that in PBMC cells (p < 0. 05). The percentage of K562 cells and PC-3 cells decreased gradually, the difference was not statistically significant (p > 0. 05), and the activity increased with the prolongation of induction time. The cytotoxicity was strongest on the 14th day after induction. After 14 days of RetroNectin stimulation, the expansion of RN-CIK cells was 330.46 鹵7.96-fold, which was significantly higher than that of normal culture methods on the 7th day, and the percentage of CD25~ cells in RN-CIK cells from the 7th day of culture was significantly higher than that of normal CIK cells (p < 0. 05). However, there was no significant difference between the two methods on the phenotype and cytotoxicity of CD3 ~ CD8 ~ + CD3 ~ CD56 ~ + CD3 ~ CD4 ~ in CIK cells (p > 0. 05). There was no significant difference in apoptosis rate between the cultured RN-CIK cells and the normal cultured CIK cells at the 11th day (p > 0. 05). The percentage of S phase cells increased gradually with the prolongation of the culture time, and reached the highest on the 7th day of culture. The percentage of S phase cells in RN-CIK cells was significantly higher than that in normal cultured CIK cells (p < 0. 05). Conclusion: (1) Anti-human CD3 monoclonal antibody, human recombinant IL-1 偽, human recombinant IFN- 緯 and human recombinant IL-2 can induce PBMC to produce CIK cells. (2) RetroNectin can significantly increase the expansion times of CIK cells. But it has little effect on phenotypic changes and cytotoxicity of CIK cells. (3) the possible mechanism of RetroNectin can promote cell proliferation: promote cell adhesion, enhance signal transduction, induce T cell activation, resist activated cell apoptosis; Promote cells from G1 phase to S phase.
【學(xué)位授予單位】：天津醫(yī)科大學(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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