【摘要】：目的:分別建立人彌漫大B細(xì)胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)阿霉素(Adriamycin,ADM)及利妥昔單抗(Rituximab,RTX)耐藥細(xì)胞株,檢測(cè)耐藥株形態(tài),增殖,藥物敏感性的變化及多藥耐藥基因1的(multidrug resistance gene 1,MDR1)表達(dá)情況,探討化療藥物與RTX之間是否存在交叉耐藥現(xiàn)象。為DLBCL耐藥研究奠定模型基礎(chǔ),對(duì)DLBCL繼發(fā)耐藥后的臨床用藥提供理論依據(jù)。方法:1.以體外低濃度梯度遞增法誘導(dǎo)構(gòu)建人耐阿霉素DLBCL細(xì)胞株P(guān)feiffer/ADM和耐RTX DLBCL細(xì)胞株P(guān)feiffer/R。2.顯微鏡下比較親代及耐藥細(xì)胞株的形態(tài)變化。3.運(yùn)用細(xì)胞計(jì)數(shù)法,繪制親代和耐藥細(xì)胞株的生長(zhǎng)曲線,計(jì)算倍增時(shí)間。4.阿霉素、依托泊苷(etoposide,VP-16)、長(zhǎng)春新堿(vincristine,VCR)對(duì)Pfeiffer、Pfeiffer/ADM、Pfeiffer/R作用72h后,運(yùn)用CCK-8法檢測(cè)細(xì)胞增殖抑制情況,計(jì)算半數(shù)抑制濃度IC50。5.以健康人血清作為補(bǔ)體來源,1μg/mL,3μg/mL,10μg/mL,30μg/mL的RTX作用于Pfeiffer、Pfeiffer/ADM、Pfeiffer/R細(xì)胞株6h后,運(yùn)用CCK-8法比較耐藥細(xì)胞株及親代細(xì)胞對(duì)RTX介導(dǎo)的補(bǔ)體依賴性細(xì)胞毒性(complement-dependent cytotoxicity,CDC)的差別。6.實(shí)時(shí)熒光定量PCR檢測(cè)Pfeiffer、Pfeiffer/ADM、Pfeiffer/R細(xì)胞株中MDR1mRNA的表達(dá)情況。結(jié)果:1.成功構(gòu)建耐ADM細(xì)胞株P(guān)feiffer/ADM及耐RTX細(xì)胞株P(guān)feiffer/R。2.顯微鏡下,Pfeiffer、Pfeiffer/ADM、Pfeiffer/R在細(xì)胞形態(tài)上無明顯差別。3.Pfeiffer,Pfeiffer/ADM,Pfeiffer/R的倍增時(shí)間分別為(32.28±1.18)h,(31.41±2.03)h及(32.03±1.77)h,三細(xì)胞株體外增殖速度相似,差異無統(tǒng)計(jì)學(xué)意義(p0.05)。4.通過cck-8法測(cè)得adm,vp16,vcr對(duì)pfeiffer的ic50值分別為(0.038±0.0090)μg/ml,(0.26±0.045)μg/ml,(0.0093±0.0023)μg/ml,對(duì)pfeiffer/adm的ic50值分別為(0.48±0.049)μg/ml,(3.42±0.45)μg/ml,(0.16±0.0032)μg/ml,計(jì)算耐藥指數(shù)分別為12.87,13.18,16.85,表明pfeiffer/adm具有多藥耐藥性。而adm,vp16,vcr對(duì)pfeiffer/r的ic50值分別為(0.038±0.046)μg/ml,(0.25±0.13)μg/ml,(0.0098±0.00014)μg/ml,與pfeiffer比較差異無統(tǒng)計(jì)學(xué)意義(p0.05),提示pfeiffer/r對(duì)化療藥物adm、vp-16和vcr依舊敏感。5.以健康人血清作為補(bǔ)體來源,1,3,10,30μg/ml的rtx作用于pfeiffer、pfeiffer/adm、pfeiffer/r細(xì)胞株6h后,對(duì)pfeiffer細(xì)胞的抑制率分別為28.58%,47.06%,69.00%,77.20%,對(duì)pfeiffer/adm為30.33%,47.21%,64.63%,72.53%,與pfeiffer細(xì)胞比較,差異無統(tǒng)計(jì)學(xué)意義(p0.05),提示pfeiffer/adm對(duì)rtx依舊敏感。pfeiffer/r細(xì)胞的抑制率分別為11.04%,24.12%,40.56%,53.58%,與pfeiffer細(xì)胞比較,差異具有統(tǒng)計(jì)學(xué)意義(p0.05),提示pfeiffer/r對(duì)rtx產(chǎn)生耐藥。6.qrt-pcr檢測(cè)發(fā)現(xiàn),pfeiffer和pfeiffer/adm細(xì)胞的mdr1mrna的相對(duì)表達(dá)量分別為(1.00±0.10)和(82033.27±4187.24),差異具有統(tǒng)計(jì)學(xué)意義(p0.001)。然而pfeiffer與pfeiffer/r細(xì)胞的mdr1mrna的相對(duì)表達(dá)量分別為(1.03±0.33)和(3.00±1.32),兩者差異無統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論:運(yùn)用低濃度梯度遞增法可成功構(gòu)建耐阿霉素人dlbcl細(xì)胞株pfeiffer/adm和耐rtx人dlbcl細(xì)胞株pfeiffer/r。pfeiffer/adm具有多藥耐藥性,其mdr1基因表達(dá)明顯上調(diào),對(duì)rtx介導(dǎo)的cdc效應(yīng)仍敏感;pfeiffer/r對(duì)rtx介導(dǎo)的cdc效應(yīng)產(chǎn)生耐藥,但對(duì)化療藥物vp-16、vcr、adm仍敏感,其mdr1基因表達(dá)無明顯變化。提示化療藥物vp-16、vcr、adm與rtx之間無交叉耐藥。本研究所構(gòu)建的耐藥細(xì)胞株是進(jìn)行下一步耐藥機(jī)制或逆轉(zhuǎn)耐藥研究的理想模型。
[Abstract]:OBJECTIVE: To establish human diffuse large B-cell lymphoma (DLBCL) adriamycin (ADM) and rituximab (RTX) resistant cell lines, detect the morphology, proliferation, changes of drug sensitivity and expression of multidrug resistance gene 1 (MDR1), and explore the chemotherapeutic drugs. Whether there is cross-resistance between substances and RTX, which lays a foundation for the study of drug resistance of DLBCL and provides a theoretical basis for clinical drug use after secondary drug resistance of DLBCL. Methods: 1. To induce the construction of human doxorubicin-resistant DLBCL cell line Pfeiffer/ADM and RTX-resistant DLBCL cell line Pfeiffer/R.2 in vitro by low concentration gradient incremental method. The growth curves of parental and drug-resistant cell lines were drawn by cell counting method, and the doubling time was calculated. 4. The inhibition of cell proliferation was detected by CCK-8 method after 72 hours of action of adriamycin, etoposide (VP-16), vincristine (VCR) on Pfeiffer, Pfeiffer/ADM and Pfeiffer/R. The inhibitory concentration IC50.5. The complement-dependent cytotoxicity (CDC) of the drug-resistant cell lines and their parental cells mediated by RTX was compared by CCK-8 assay after 6 hours of treatment of Pfeiffer, Pfeiffer/ADM, Pfeiffer/R cell lines with healthy human serum as complement source, 1 ug/mL, 3 ug/mL, 10 ug/mL, 30 ug/mL RTX. The expression of MDR1 mRNA in Pfeiffer, Pfeiffer/ADM and Pfeiffer/R cell lines was detected by fluorescence quantitative PCR. Results: 1. ADM-resistant cell lines Pfeiffer/ADM and RTX-resistant cell lines Pfeiffer/R.2 were successfully constructed. Under microscope, Pfeiffer, Pfeiffer/ADM, Pfeiffer/R had no significant difference in cell morphology. The proliferation rates of the three cell lines in vitro were similar, but there was no significant difference (p0.05). 4. the IC50 values of adm, VP16 and VCR to Pfeiffer were (0.038 + 0.0090) UG / ml, (0.26 + 0.045) UG / ml, (0.0093 + 0.0023) UG / ml, and (0.04 + 0.04) icfeiffer / ADM by cck-8, respectively. 9) UG / ml, (3.42 (+ 0.45) UG / ml, (0.16 (+ 0.0032) UG / ml, and the calculated drug resistance indices were 12.87, 13.18, 16.85, respectively, indicating that Pfeiffer / ADM had multidrug resistance, while the IC50 values of adm, vp16, VCR for Pfeiffer / r were (0.038 (+ 0.046) UG / ml, (0.25 (+ 0.13) UG / ml, (0.0098 (+ 0.00014) UG / ml, respectively, which had no significant difference with Pfeiffer (p0.05). Pfeiffer / R was still sensitive to adm, VP-16 and vcr. 5. the inhibition rates of Pfeiffer / R cells were 28.58%, 47.06%, 69.00%, 77.20%, 30.21%, 64.63% and 72.53% respectively, after 6 hours of Pfeiffer / adm, Pfeiffer / ADM and Pfeiffer / R cells were treated with 1,3,10,30 UG / ml RTX from healthy human serum as a complement source. The inhibition rates of Pfeiffer / R cells were 11.04%, 24.12%, 40.56%, 53.58% respectively. the difference was statistically significant compared with Pfeiffer cells (p0.05), suggesting that Pfeiffer / R had resistance to rtx. 6.qrt-pcr detection showed that Pfeiffer and Pfeiffer / ADM cells were still sensitive to MDR1 mrn. The relative expression of a was (1.00 0.10) and (82033.27 4187.24), respectively. however, the relative expression of MDR1 mRNA in Pfeiffer and Pfeiffer / R cells was (1.03 0.33) and (3.00 1.32), respectively. there was no significant difference between them (p0.05). conclusion: the low concentration gradient method can be successfully used to construct a drug-resistant strain. Human DLBCL cell lines pfeiffer/adm and rtx-resistant human DLBCL cell lines pfeiffer/r.pfeiffer/adm have multidrug resistance, and their MDR1 gene expression is significantly up-regulated, and they are still sensitive to rtx-mediated CDC effect; pfeiffer/r is resistant to rtx-mediated CDC effect, but still sensitive to chemotherapy drugs vp-16, VCR and adm, and their MDR1 gene expression is not significantly changed. There is no cross-resistance between chemotherapy drugs vp-16, vcr, ADM and rtx. The drug-resistant cell lines constructed in this study are ideal models for further study of drug resistance mechanism or reversal of drug resistance.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.1

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