【摘要】：第一章：鼻咽癌細(xì)胞株CNE-2Z中腫瘤干細(xì)胞樣細(xì)胞的懸浮培養(yǎng)和鑒定目的：探討富集鼻咽癌腫瘤干細(xì)胞樣細(xì)胞的方法并鑒定鼻咽癌細(xì)胞株CNE-2Z中的腫瘤干細(xì)胞樣細(xì)胞。方法：(1)取鼻咽癌細(xì)胞株CNE-2Z普通培養(yǎng)至對(duì)數(shù)生長期的細(xì)胞,消化后分別細(xì)胞計(jì)數(shù),密度達(dá)到1×103/ml.1×104/m1、1×105/ml、1×106 ／ml時(shí),分別加入無血清培養(yǎng)液中繼續(xù)培養(yǎng),以0.3%臺(tái)盼藍(lán)計(jì)數(shù)第0、1、3、5、7、9天活細(xì)胞百分率；(2)制備普通培養(yǎng)活細(xì)胞和第9天無血清培養(yǎng)活細(xì)胞爬片,用CD133和CD44人抗鼠單克隆抗體免疫組化染色；(3)用第9天無血清培養(yǎng)活細(xì)胞制備電鏡觀察標(biāo)本并進(jìn)行電鏡觀察。結(jié)果：(1)細(xì)胞密度為1×103／ml.1×10/ml.1×105/ml.1×106/ml時(shí)的細(xì)胞懸液再經(jīng)無血清懸浮培養(yǎng)至第9天時(shí),其中的活細(xì)胞百分率分別是0.00%、0.05%、3.10%、0.27%,以1 x1 05/ml組活細(xì)胞比率最高；(2)普通培養(yǎng)細(xì)胞爬片的CD133和CD44細(xì)胞強(qiáng)陽性率分別是2.71%和3.00%,而無血清培養(yǎng)細(xì)胞爬片的CD133和CD44強(qiáng)陽性率分別為66.7%和67.00%,兩種培養(yǎng)方法強(qiáng)陽性細(xì)胞比較,差異有非常顯著性統(tǒng)計(jì)學(xué)意義(P0.01),CD133和CD44在兩種培養(yǎng)液中強(qiáng)陽性細(xì)胞百分率相關(guān)性分析呈正相關(guān)(P0.01)；(3)掃描電鏡和透射電鏡觀察所見CNE-2Z鼻咽癌細(xì)胞中腫瘤干細(xì)胞樣細(xì)胞表面有鱗片樣改變,伴有出芽樣突起,透射電鏡下,細(xì)胞呈現(xiàn)較大異質(zhì)性,可為圓形或橢圓形,細(xì)胞核固縮,細(xì)胞質(zhì)極度濃縮和細(xì)胞器嚴(yán)重減少,可見類凋亡(?)小體以類似出胞的方式排出胞外。結(jié)論：1.無血清懸浮培養(yǎng)法可富集CNE-2Z鼻咽癌細(xì)胞中的腫瘤干細(xì)胞樣細(xì)胞,1×105/ml的細(xì)胞濃度是CNE-2Z鼻咽癌細(xì)胞的最佳懸浮培養(yǎng)細(xì)胞密度。無血清懸浮培養(yǎng)法是一種比較經(jīng)濟(jì)、易于掌握的培養(yǎng)腫瘤干細(xì)胞的較好方法,值得推廣應(yīng)用。2.CD1 33和CD44可作為鼻咽癌腫瘤干細(xì)胞樣細(xì)胞的表面標(biāo)志物而應(yīng)用于鼻咽癌腫瘤干細(xì)胞樣細(xì)胞的檢測(cè)；在無血清懸浮培養(yǎng)液中,鼻咽癌腫瘤干細(xì)胞樣細(xì)胞以自我復(fù)制和增殖兩種方式分裂生長,但生長緩慢。3.電子顯微鏡下,鼻咽癌腫瘤干細(xì)胞樣細(xì)胞的形態(tài)特征有較大異質(zhì)性,細(xì)胞核固縮,細(xì)胞質(zhì)極度濃縮和細(xì)胞器嚴(yán)重減少,細(xì)胞質(zhì)中可見類凋亡(?)小體以類似出胞的方式排出胞外,這些變化可能與無血清環(huán)境改變了細(xì)胞的生物學(xué)特性有關(guān)。第二章：鼻咽癌細(xì)胞株CNE-2Z腫瘤干細(xì)胞樣細(xì)胞的生物學(xué)特性及益氣解毒方對(duì)其分化潛能的干預(yù)效應(yīng)目的：研究鼻咽癌細(xì)胞株CNE-2Z中腫瘤干細(xì)胞樣細(xì)胞的生物學(xué)特性及益氣解毒方對(duì)該類細(xì)胞分化潛能的干預(yù)效應(yīng)。方法：(1)采用無血清懸浮培養(yǎng)方法富集CNE-2Z鼻咽癌細(xì)胞中的腫瘤干細(xì)胞樣細(xì)胞球；(2)分別采用無血清懸浮培養(yǎng)轉(zhuǎn)含胎牛血清的普通培養(yǎng)法及持續(xù)無血清培養(yǎng)法檢測(cè)培養(yǎng)體系中懸浮細(xì)胞球的克隆形成能力、分化增殖能力,并以不同密度的細(xì)胞球懸液注射至裸鼠皮下,觀察其成瘤能力；(3)用益氣解毒方含藥血清配制的溫育液與無血清懸浮培養(yǎng)所得腫瘤干細(xì)胞樣細(xì)胞球共培養(yǎng),分別設(shè)立普通組和空白對(duì)照組,比較24 hrs、72 hrs、5天等時(shí)間段時(shí)細(xì)胞球中CD133/CD44陽性細(xì)胞數(shù)的變化。結(jié)果：(1)將培養(yǎng)體系中獲得的CNE-2Z鼻咽癌細(xì)胞球吹散后再行無血清懸浮培養(yǎng),仍然可形成細(xì)胞球；將這樣的細(xì)胞球轉(zhuǎn)至普通培養(yǎng),24 hrs后可見在培養(yǎng)體系中有單個(gè)細(xì)胞貼壁生長,7天后細(xì)胞球消失；貼壁生的細(xì)胞的形態(tài)特征與原親代細(xì)胞無異；對(duì)這些貼壁細(xì)胞的免疫組化檢測(cè)顯示CD133+/CD44+細(xì)胞陽性率逐日下降,各時(shí)段比較均有統(tǒng)計(jì)學(xué)意義(P0.05)；交替培養(yǎng)時(shí),目標(biāo)細(xì)胞在細(xì)胞球和貼壁細(xì)胞之間交替出現(xiàn),細(xì)胞(球)形態(tài)無明顯改變；(2)無血清懸浮培養(yǎng)細(xì)胞球經(jīng)洗滌后,分別以1×103、1×105、1×107的細(xì)胞密度進(jìn)行裸鼠皮下注射,均可形成移植瘤,腫瘤生長速度與注射的細(xì)胞密度有關(guān)；移植瘤原代培養(yǎng)細(xì)胞生長速度明顯快于其來源的親代細(xì)胞株細(xì)胞(P0.05)；(3)益氣解毒方含藥血清溫育液中CD133/CD44陽性細(xì)胞下降速率明顯快于普通培養(yǎng)組和對(duì)照組,各時(shí)段與普通組、對(duì)照組比較均有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：1、無血清懸浮培養(yǎng)法所得CNE-2Z鼻咽癌細(xì)胞球具有自我更新、高速分化增殖和高效體內(nèi)成瘤等腫瘤干細(xì)胞特性,可能為其中的腫瘤干細(xì)胞樣細(xì)胞群。2、益氣解毒方體外作用對(duì)CNE-2Z鼻咽癌細(xì)胞中的腫瘤干細(xì)胞樣細(xì)胞具有明顯的分化誘導(dǎo)效應(yīng)。第三章：益氣解毒方對(duì)CNE-2Z鼻咽癌細(xì)胞裸鼠移植瘤中腫瘤干細(xì)胞樣細(xì)胞干預(yù)作用的初步研究目的：探討益氣解毒方對(duì)CNE-2Z鼻咽癌細(xì)胞裸鼠移植瘤組織中腫瘤干細(xì)胞樣細(xì)胞的干預(yù)效應(yīng),比較不同給藥方案對(duì)這類腫瘤干細(xì)胞樣細(xì)胞的干預(yù)效率以及對(duì)裸鼠生存質(zhì)量的影響。方法：建立CNE-2Z鼻咽癌細(xì)胞裸鼠移植瘤模型；隨機(jī)分為模型組、中藥組(益氣解毒方組)、DDP組(順鉑)和聯(lián)合用藥組(益氣解毒方+DDP),分別給予生理鹽水、益氣解毒方藥液、順鉑單藥和益氣解毒方藥液+順鉑處理；觀察用藥前后各組移植瘤裸鼠體重、皮膚顏色、移植瘤瘤體大小、一般生活狀態(tài)等相關(guān)指標(biāo)變化；用藥2周后獲取移植瘤瘤組織標(biāo)本,免疫組織化學(xué)染色法測(cè)定并分析比較各組移植瘤組織細(xì)胞CD133和CD44表達(dá)情況,western blotting法檢測(cè)各組移植瘤組織CD133和CD44蛋白含量。結(jié)果：1)四組移植瘤裸鼠用藥前后的體重變化表現(xiàn)為：模型組和益氣解毒方組用藥前后體重比較有非常顯著性差異(P0.01),聯(lián)合用藥組和DDP組用藥前后體重比較無顯著性差異(P0.05)；移植瘤瘤體大小變化表現(xiàn)為：益氣解毒方組、聯(lián)合用藥組、DDP組三組與模型組比較均有顯著性差異(P0.05),益氣解毒方組、聯(lián)合用藥組、DDP組抑瘤率依次為18.7%、58.4%、43.8%；皮膚顏色和生活狀態(tài)變化表現(xiàn)為：模型組和益氣解毒方組用藥前后無明顯差異,聯(lián)合用藥組和DDP組用藥前后有顯著性差異,即皮膚顏色變暗,生活活力下降,尤其DDP組明顯。2)四組裸鼠移植瘤中CD133/CD44蛋白質(zhì)的western blotting檢測(cè)結(jié)果為：在Mark條帶120 kDa.80-95 kDa分子量附近均出現(xiàn)相應(yīng)蛋白質(zhì)條帶。但各組移植瘤標(biāo)本的相應(yīng)蛋白質(zhì)條帶亮度有差異。模型組最亮,益氣解毒方組和DDP組較亮,聯(lián)合用藥組最暗。聯(lián)合用藥組與DDP組、益氣解毒方組、模型組比較亮度差異有顯著性。3)四組裸鼠移植瘤組織中CD133和CD44免疫組化檢測(cè)結(jié)果：CD133的表達(dá)為各用藥組與模型組比較均有顯著性差異(P0.05),益氣解毒方組、DDP組與聯(lián)合用藥組比較有顯著性差異(P0.05),益氣解毒方組與DDP組比較無顯著性差異(P0.05)；CD44的表達(dá)結(jié)果與CD133相近,即各用藥組與模型組比較均有顯著性差異(P0.05)；益氣解毒方組、DDP組與聯(lián)合用藥組比較有顯著性差異(P0.05)；益氣解毒方組與DDP組比較無顯著性差異(P0.05)；CD133和CD44比較,除聯(lián)合用藥組外,各組CD44陽性表達(dá)率明顯高于CD133,二者相關(guān)性分析表現(xiàn)為高度正相關(guān)(P0.01)。結(jié)論1.CNE-2Z鼻咽癌細(xì)胞移植瘤組織中存在少量鼻咽癌腫瘤干細(xì)胞樣細(xì)胞,且CD44陽性細(xì)胞表達(dá)率高于CD133；作為鼻咽癌腫瘤干細(xì)胞樣細(xì)胞的表面標(biāo)志物而言,二項(xiàng)指標(biāo)中究竟哪一項(xiàng)具有更高的特異性,有待進(jìn)一步闡明。2、順鉑單藥組中CD133/CD44陽性細(xì)胞表達(dá)率明顯高于聯(lián)合用藥組,提示抗癌藥物順鉑可能有富集鼻咽癌腫瘤干細(xì)胞樣細(xì)胞的作用；而中藥益氣解毒方可能對(duì)鼻咽癌腫瘤干細(xì)胞樣細(xì)胞具有誘導(dǎo)分化作用。3、在幾種給藥方案中,單純使用中藥的抑瘤能力有限,單純使用化療藥則對(duì)荷瘤機(jī)體損害較大,中西醫(yī)結(jié)合用藥方案則在提高宿主生存質(zhì)量、有效殺滅癌細(xì)胞乃至腫瘤干細(xì)胞方面具有協(xié)同作用,不啻為一種更為合理的用藥選擇。
[Abstract]:Chapter 1: Suspension culture and identification of tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z Objective: To explore the method of enriching tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z and identify tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z. When the cell density reached 1 103/ml.1 65 Results: (1) When the cell density was 1 x 103/ml.1 x 10/ml.1 x 105/ml.1 x 106/ml, the percentage of viable cells was 0.00%, 0.05%, 3.10%, 0.27%, respectively, when the cell suspension was cultured in serum-free medium for 9 days. (2) The strong positive rates of CD133 and CD44 cells were 2.71% and 3.00% respectively in common culture slices, 66.7% and 67.00% in serum-free culture slices, respectively. The difference between the two culture methods was statistically significant (P 0.01). CD133 and CD44 were in two groups. There was a positive correlation between the percentage of strong positive cells in the culture medium (P 0.01); (3) Scanning electron microscopy and transmission electron microscopy (TEM) showed that the surface of tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma cells showed squamous-like changes, accompanied by budding protuberances. Under transmission electron microscopy, the cells showed large heterogeneity, which could be round or oval, and the nucleus of the cells was pyknosis. Conclusion: 1. Serum-free suspension culture can enrich tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma cells, and the best suspension culture cell density is 1 X105 / ml. CD1 33 and CD44 can be used as the surface markers of nasopharyngeal carcinoma tumor stem cell-like cells for detection of nasopharyngeal carcinoma tumor stem cell-like cells; in serum-free suspension medium, nasopharyngeal carcinoma tumor stem cell-like cells are fine. Cells divide and grow in two ways of self-replication and proliferation, but grow slowly. Chapter 2: Biological characteristics of nasopharyngeal carcinoma cell line CNE-2Z and the intervention effect of Yiqi Jiedu Recipe on its differentiation potential Objective: To study the biological characteristics of tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z and the effects of Yiqi Jiedu Recipe on its differentiation potential. Methods: (1) Tumor stem cell-like cell spheres in CNE-2Z nasopharyngeal carcinoma cells were enriched by serum-free suspension culture method; (2) The cloning of suspension cell spheres was detected by serum-free suspension culture and continuous serum-free culture respectively. The ability of formation, differentiation and proliferation were observed by injecting different density of cell suspension into nude mice subcutaneously. (3) The tumor-forming ability was observed by co-culture of tumor stem cell-like cell spheres obtained from serum containing Yiqi Jiedu recipe and serum-free suspension culture. Results: (1) The CNE-2Z nasopharyngeal carcinoma cell spheres obtained in the culture system were blown away and then cultured in serum-free suspension, and the spheres could still form cell spheres. When the spheres were transferred to normal culture, a single cell adhered to the wall and grew fine after 7 days. The positive rate of CD133 + / CD44 + cells decreased day by day, and there was statistical significance (P 0.05). In the alternate culture, the target cells appeared alternately between the cell sphere and adherent cells, and the cells (spheres) were shaped. (2) Subcutaneous injection of serum-free suspension cultured cell spheres at the cell densities of 1 x 103, 1 x 105, and 1 x 107 after washing resulted in the formation of transplanted tumors, the growth rate of which was related to the density of injected cells, and the growth rate of primary cultured cells was significantly faster than that of parental cells (P 0.05). (3) The decrease rate of CD133/CD44 positive cells in serum culture medium containing Yiqi Jiedu Recipe was significantly faster than that in normal culture group and control group, and there was statistical significance (P 0.05) in comparison with normal group and control group. Tumor stem cell characteristics such as in vivo tumorigenesis, may be one of the tumor stem cell-like cells. 2, Yiqi Jiedu Recipe has obvious differentiation induction effect on CNE-2Z nasopharyngeal carcinoma cells in vitro. Chapter 3: Yiqi Jiedu Recipe on CNE-2Z nasopharyngeal carcinoma cells transplanted in nude mice tumor stem cell-like cells intervention Objective: To investigate the intervention effect of Yiqi Jiedu Recipe on tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma xenografts in nude mice, and compare the intervention efficiency of different dosage regimens on these tumor stem cell-like cells and the quality of life in nude mice. The model was randomly divided into model group, Chinese medicine group (Yiqi Jiedu Fang group), DDP group (cisplatin) and combined treatment group (Yiqi Jiedu Fang + DDP), respectively, given saline, Yiqi Jiedu Fang liquid, cisplatin single drug and Yiqi Jiedu Fang liquid + cisplatin treatment; observed the weight of transplanted tumor, skin color, tumor size, 1. The expression of CD133 and CD44 in transplanted tumor tissues was detected by immunohistochemical staining, and the contents of CD133 and CD44 protein in transplanted tumor tissues were detected by Western blotting. Results: 1) The four groups of transplanted tumor tissues were detected before and after treatment. Weight changes showed: the model group and Yiqi Jiedu Fang before and after the treatment of body weight compared with a very significant difference (P 0.01), combined treatment group and DDP group before and after the weight of no significant difference (P 0.05); transplanted tumor size changes showed: Yiqi Jiedu Fang group, combined treatment group, DDP group compared with the model group were three groups. Significant difference (P 0.05), Yiqi Jiedu Prescription group, combined drug group, DDP group tumor inhibition rate was 18.7%, 58.4%, 43.8%; skin color and life state changes showed: model group and Yiqi Jiedu Prescription group before and after treatment, no significant difference, combined drug group and DDP group before and after treatment, that is, skin color darkening, life vitality. The Western blotting results of CD133/CD44 protein in four groups of nude mice transplanted tumor showed that there were corresponding protein bands near the molecular weight of Mark band 120 kDa.80-95 kDa. CD13 3 and CD44 immunohistochemical results in the transplanted tumor tissues of the four groups: the expression of CD13 3 in each group was significantly different from that in the model group (P 0.05). The expression of CD13 3 was significantly different between the treatment group and the model group (P 0.05). There was significant difference (P 0.05), there was no significant difference between Yiqi Jiedu Prescription group and DDP group (P 0.05); the expression of CD44 was similar to that of CD133, that is, there was significant difference between each drug group and model group (P 0.05); Yiqi Jiedu Prescription group, DDP group and combined drug group had significant difference (P 0.05); Yiqi Jiedu Prescription group and DDP group had no significant difference (P 0.05); Compared with CD133 and CD44, the positive expression rate of CD44 in each group was significantly higher than that of CD133, and the correlation analysis showed a high positive correlation (P 0.01). As a surface marker of nasopharyngeal carcinoma stem cell-like cells, which of the two indicators has higher specificity remains to be further clarified. 2. The expression rate of CD133/CD44 positive cells in cisplatin monotherapy group is significantly higher than that in combination therapy group, suggesting that cisplatin may enrich nasopharyngeal carcinoma stem cell-like cells. Yiqi Jiedu Decoction may induce differentiation of nasopharyngeal carcinoma stem cell-like cells. 3. In several drug regimens, the inhibition ability of traditional Chinese medicine alone is limited, while chemotherapy alone is harmful to the tumor-bearing organism. The combination of traditional Chinese and Western medicine regimen can improve the quality of life of the host and kill cancer cells effectively. The synergistic effect on cancer stem cells is a more rational choice.
【學(xué)位授予單位】：湖南中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63

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1 高霞;中藥蘇木和新合成的化學(xué)藥對(duì)血源性細(xì)胞的誘導(dǎo)作用研究[D];上海師范大學(xué);2008年

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