腫瘤壞死因子-α(rhTNF-α)對(duì)人食管癌細(xì)胞TE13、KYSE140和SEG1的放療增敏實(shí)驗(yàn)研究.pdf

1、葛宜枝rhrrNF0【對(duì)人食管癌細(xì)胞TEl3、KYSEl40和SEGI的放療增敏實(shí)驗(yàn)研究中文摘要目的：觀察腫瘤壞死因子僅(rhTNFa)聯(lián)合X射線(xiàn)對(duì)三種不同分化程度的人食管癌細(xì)胞株TE13(低分化鱗癌)、KYSE140(中分化鱗癌)和SEG1(高分化腺癌)的生長(zhǎng)抑制及放射治療中的增敏效應(yīng)，探討TNF0【聯(lián)合X射線(xiàn)治療對(duì)食管癌放療增敏作用的機(jī)制，為其在食管癌臨床治療的應(yīng)用提供一定的理論與實(shí)驗(yàn)基礎(chǔ)。方法：三種不同分化程度的人食管癌細(xì)胞株(T

2、E13，KYSE140和SEG1)培養(yǎng)于含10％胎牛血清的RMPI1640培養(yǎng)基中。rhTNF0【體外作用于這三種食管癌細(xì)胞株，藥物濃度分別為100IU／ml，200IU／ml，400IU／ml，1200IU／ml，1600IU／ml，3200IU／ml六個(gè)梯度，根據(jù)公式計(jì)算出IC50，采用此濃度作為實(shí)驗(yàn)作用濃度。將培養(yǎng)至對(duì)數(shù)生長(zhǎng)期的三種人食管癌細(xì)胞分別分為四組：對(duì)照組，單獨(dú)rhTNF儀作用組，單獨(dú)X射線(xiàn)照射作用組和rhTNF—Gt聯(lián)合

3、x射線(xiàn)照射作用組，分別培養(yǎng)12h，24h，48h，72h，然后采用MTT法檢測(cè)吸光度(OD值)，根據(jù)結(jié)果繪制細(xì)胞生長(zhǎng)曲線(xiàn)。采用同樣的分組方法，將以上三種人食管癌細(xì)胞株培養(yǎng)24h，經(jīng)處理后用流式細(xì)胞儀分別檢測(cè)上述三種細(xì)胞各組的凋亡情況。用克隆形成試驗(yàn)檢測(cè)經(jīng)rhTNFa處理后三種細(xì)胞的細(xì)胞存活分?jǐn)?shù)和克隆形成率，并計(jì)算放療增敏比。通過(guò)Westernblot方法分別檢測(cè)三種人食管癌細(xì)胞對(duì)照組和實(shí)驗(yàn)組的放療增敏相關(guān)蛋白NFrd3和凋亡相關(guān)蛋白Ca

4、spase3的表達(dá)效應(yīng)。結(jié)果：1、細(xì)胞生長(zhǎng)抑制效應(yīng)：細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示，TE13，KYSE140和SEG1三種人食管癌細(xì)胞株的半數(shù)致死濃度分別為400IU／ml，400IU／ml，1200IU／ml。MTT實(shí)驗(yàn)結(jié)果表明，半數(shù)致死濃度的rhTNFu處理后，各組細(xì)胞的抑制率隨時(shí)間的增加而增加，24h后抑制程度逐漸變緩。rhTNF0t對(duì)這三種細(xì)胞生長(zhǎng)有一定的抑制作用，但統(tǒng)計(jì)學(xué)處理沒(méi)有意義，而rhTNFc【聯(lián)合X射線(xiàn)照射作用組，TE13，KY

5、SE140和SEG1三種細(xì)胞的生長(zhǎng)抑制抑制率為03880026，O5360016，02630061，分別與其對(duì)應(yīng)的對(duì)照組(01290086，0140a：0086，00960066)、單獨(dú)rhTNF0【作用組(02140126，02090024，O1290077)和單獨(dú)X射線(xiàn)照射作用組(01580109，03140099，01770109)相比有顯著差異(P005)。2、流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡效應(yīng)：?jiǎn)为?dú)rhTNF0【作用組、單獨(dú)X射線(xiàn)照射

6、作用組的葛宜枝rhTNF—c【對(duì)人食管癌細(xì)胞TEl3、KYSEl40和SEGl的放療增敏實(shí)驗(yàn)研究3Purpose：AbstractToassesstheeffectsoftumornecrosisfactor0【(TNF0t)onthedegreeofdifferentiationofthreekindsofesophagealcancercelllines，TE13(poorlydifferentiatedsquamouscellca

7、rcinomas)，KYSE140(moderatelydifferentiatedsquamouscellcarcinomas)andSEG1(adenocarcinoma)，thecellgrowthinhibitionandtheradiotherapysensitizationeffectinvitroMethods：Threedifferentdifferentiationdegreesofesophagealcancerce

8、lllines，TE13，KYSE140andSEG1wereculturedinthemediumcontaining10％fetalbovineserumRMPI1640rhTNF一(Itwererespectivelyactedontheseesophagealcancercelllines(100IU／ml，200IU／ml，400IU／ml，1200IU／ml，1600IU／ml，3200IU／m1)，IC50(halfmax

9、imalinhibitoryconcentration)werecalculatedaccordingtothecorrespondingregressionequmion，whichwereadoptedasexperimentalconcentrationAllthreecelllinesweredividedintocontrolgroup，onlyrhTNF—c【treatedgroup，onlyXrayradiationgro

10、upandcombinedgroupwhentheydevelopedtothelogarithmicphaseAbsorbancevalue(ODvalue)weredetectedafter12h，24h，48h，72hThenthecellgrowthinhibitioncurvecanbedrawnthroughMTTmethodAbovementionedgroupingmethodstillbeadopted，flowcyt

11、ometrywasusedtodetectcellapoptosisafter24h，cloneformationtestwasaimedatradiotherapysensitizationandWesternblotmethodwasconsideredtodetecttheexpressionofapoptosisrelatedgeneperformedbythreeesophagealcancercelllinesResults

12、：1Cellgrowthinhibitioncurve：ApoptosiscalvesofthreekindsofesophagealcancercelllinesthoseweretreatedbyrhTNF僅withdifferentconcentrationsshowedthatIC50wereapartly400IU／ml，400IU／ml，and1200IU／m1Undertheseconcentrations，thecell

13、s’inhibitionrateincreasedovertimeandslowedafter24hrhTNF0【hasinhibitoryeffectamongthreecelllinesandthestrongestiscombinedgroup，whichisdistinctlyhigherthanotherthreegroups2Flowcytometrytodetectcellapoptosis：Theapoptosisrat