【摘要】： 目的：初步研究RetroNectin對CIK細胞增值、表性變化及殺傷活性的影響，并探討其可能機制。 方法：采集人外周血單個核細胞，標本分為兩組，對照組加入IFN-γ、IL-2、IL-1α、CD3mAb等細胞因子誘導其增殖，實驗組另外加入RetroNectin誘導，倒置顯微鏡下觀察培養(yǎng)過程中細胞形態(tài)變化，采用活細胞計數(shù)法觀察CIK細胞增殖，分別繪制細胞增值曲線；流式細胞術檢測CIK細胞培養(yǎng)過程中的表型變化；LDH法檢測CIK細胞對腫瘤細胞K562細胞和PC-3細胞的殺傷活性；采用AnnexinV／PI染色，，流式細胞術檢測細胞凋亡情況；流式細胞技術檢測不同培養(yǎng)條件下第4d、7d、11d及14d的CIK細胞周期。 結果：PBMC在體外經(jīng)過多種細胞因子的誘導后，能大量擴增生成CIK細胞，培養(yǎng)14天，擴增倍數(shù)可達96.13±5.03倍；CIK細胞中的CD3~+CD8~+、CD3~+CD56~+細胞比例隨培養(yǎng)時間的延長較PBMC明顯增多(p＜0.05)；細胞的百分率則逐漸下降；CIK細胞對K562細胞和PC-3細胞均有較強的殺傷活性，兩者相比較，差異無統(tǒng)計學意義(p＞0.05)，并且隨著誘導時間的延長，這種殺傷活性增強，在誘導培養(yǎng)的第14d殺傷活性最強。經(jīng)RetroNectin刺激14天后的RN-CIK細胞擴增倍數(shù)可達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細胞中的CD25~+細胞比例較普通培養(yǎng)的CIK細胞明顯增多(p＜0.05)。但兩種培養(yǎng)方法對CIK細胞的CD3~+CD8~+、CD3~+CD56~+、CD3~+CD4~+表型及細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細胞與普通培養(yǎng)的CIK細胞凋亡率無明顯差異(p＞0.05)。S期細胞的比例隨所培養(yǎng)時間的延長逐漸升高，在培養(yǎng)的第7d達到最高，并且RN-CIK細胞中S期的細胞比例明顯高于普通培養(yǎng)的CIK細胞組(p＜0.05)。 結論：(1)體外應用抗人CD3單抗、人重組IL-1α、人重組IFN-γ和人重組IL-2能誘導PBMC生成CIK細胞，CIK細胞對K562細胞和PC-3細胞均有較強的殺傷活性。(2)RetroNectin可以明顯增加CIK細胞的擴增倍數(shù)，但是對CIK細胞的表型變化及殺傷活性影響不大。(3)RetroNectin能促進細胞增殖的可能機制：促進細胞之間的黏附，增強信號傳導；誘導T細胞活化；抵抗活化細胞凋亡；促使細胞由G1期進入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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

【參考文獻】

相關期刊論文 前7條

1 任歡,邢淑賢,周貴生,王立榮,田景先,李殿俊;CIK細胞與LAK細胞對H-22腹水瘤的抑瘤作用[J];哈爾濱醫(yī)科大學學報;2000年02期

2 王裕發(fā);陳霞芳;巫協(xié)寧;;可溶性白介素-2受體在腫瘤診斷與治療中的價值[J];上海醫(yī)學;1993年02期

3 孫紅,姚珍;TGF-β與細胞周期調節(jié)[J];生命科學;1998年02期

4 童春容,陸道培,丘鏡瑩,何萁;細胞因子誘導的殺傷細胞對慢性髓性白血病細胞的體外凈化作用[J];實驗血液學雜志;1996年03期

5 馬建輝，陳毓仙，許秉責，石衛(wèi)，李艷芬;過繼TAK細胞免疫治療在晚期腎癌的應用價值[J];中華泌尿外科雜志;1995年12期

6 施明,張冰,湯紫榮,雷周云,王慧芬,馮永毅,劉敬超,范振平,李捍衛(wèi),牟勁松,王福生;肝癌患者自體細胞因子誘導殺傷細胞治療后免疫活性細胞的檢測及其臨床意義[J];中華醫(yī)學雜志;2003年23期

7 于津浦,任秀寶,張澎,安秀梅,張乃寧,郝希山;惡性實體瘤患者自體CIK細胞的體外大量擴增與生物學指標檢測[J];中國腫瘤生物治療雜志;2001年03期

本文編號：2192365