肝再生增强因子对多发性骨髓瘤细胞增殖的影响及其机制研究
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摘要
背景：多发性骨髓瘤（multiple myeloma，MM）是一种来源于浆细胞的血液系统恶性肿瘤，它的发生率在血液系统肿瘤中居第二位[1]。MM的发病机制尚不清楚，目前骨髓微环境中细胞因子与骨髓瘤生长的关系是研究的热点，已发现白细胞介素-6（IL-6）、肿瘤坏死因子(TNF)、肝细胞生长因子(HGF)、血管内皮生长因子(VEGF)、白细胞介素-10（IL-10）、白细胞介素-21（IL-21）等细胞因子在骨髓瘤细胞的增殖、抗凋亡和耐药性产生等方面起了很重要的作用[2-5],它们通过作用于特异的受体再活化JAK/STAT3、PI-3激酶/AKT、Ras/MAPK、NF-κB、β-catenin旁路等几种信号转导通路，导致骨髓瘤细胞增殖。
我们课题组在利用BALB/c小鼠的骨髓瘤细胞SP2/0和受免疫BALB/c小鼠的B淋巴细胞融合而成的杂交瘤细胞制备抗人肝再生增强因子（Augmenter of Liver Regeneration,ALR）单抗时偶然发现：将能产生抗人ALR单抗的杂交瘤细胞注射入BALB/c小鼠腹腔内，从而产生含高浓度抗人ALR单抗的腹水，我们进行了多次制备，每次五只小鼠，然而让人惊奇的是，每次总有2-3只小鼠在产生1-2轮腹水后，腹水自行消失，开腹检查找不到任何接种杂交瘤细胞的痕迹。由此我们推测，骨髓瘤细胞可能产生和分泌大量ALR，并且严重依赖ALR来维持其恶性生长，由于小鼠的B淋巴细胞产生的抗人ALR单抗中和了骨髓瘤细胞增殖依赖的ALR，导致骨髓瘤细胞缺乏维持其恶性生长的刺激信号而发生凋亡，最终腹水自行消失。
肝再生增强因子是上世纪90年代初被克隆的一种新的热稳定性、非特异性促进损伤肝脏修复的细胞因子，分子量约为15KD，其氨基酸序列和分子结构均不同于肝细胞生长因子（HGF），它在体内多种组织器官中有表达[6]。目前对它的生物学作用研究主要局限于肝脏。我们课题组前期实验发现，ALR在体外能刺激肝癌细胞的增殖，且其促增殖效应与ALR的浓度成正相关，但是ALR对原代肝细胞的增殖无刺激作用[7、8]。实验证明ALR在肝癌组织中是高表达的，而正常成人大多数肝细胞均不表达ALR[9、10]，这些都提示ALR在肝癌的发生、发展中起了重要作用。我们课题组最新的研究（基金资助项目：30570826）也证明用ALR特异性单抗阻断肝癌细胞分泌的ALR与其受体结合或用siRNA在转录水平阻止ALR表达，均能明显抑制肝癌细胞增殖和移植瘤的生长[11、12]。
关于ALR在多发性骨髓瘤中的表达及其在多发性骨髓瘤增殖中的作用，目前国内外尚无任何研究报道。因此，我们以此为切入点，针对以上ALR是否参与多发性骨髓瘤发生、发展的作用进行研究，以期阐明ALR在多发性骨髓瘤发生、发展中的作用和机制，并为多发性骨髓瘤的治疗提供新的参考靶点和实验依据。
本研究主要分为三个部分：
第一部分：ALR在人多发性骨髓瘤细胞株U266中的表达研究。
目的：通过对人多发性骨髓瘤细胞株U266中ALR的mRNA和蛋白水平的检测，证明多发性骨髓瘤细胞高表达ALR，为进一步探讨ALR在多发性骨髓瘤细胞增殖中的作用奠定基础。
方法：利用Realtime PCR检测人多发性骨髓瘤细胞株U266、小鼠骨髓瘤细胞株SP2/0及正常人外周血单个核细胞PMBC中ALR的mRNA表达水平；利用Western-blot检测U266、SP2/0及PMBC细胞中ALR的蛋白表达水平。
结果：Realtime PCR结果显示人多发性骨髓瘤细胞株U266和小鼠骨髓瘤细胞株SP2/0中ALR的mRNA表达水平明显高于正常人外周血单个核细胞PMBC。Western-blot结果显示U266、SP2/0细胞株中ALR蛋白表达水平较高，而PMBC组未见ALR蛋白表达。
结论：在多发性骨髓瘤细胞株U266中，ALR mRNA及蛋白水平均为高表达。
第二部分：ALR对人多发性骨髓瘤细胞株U266增殖及凋亡的影响研究。
目的：检测外源性加入ALR及抗ALR单克隆抗体后U266细胞增殖情况；构建表达ALR基因的shRNA干扰慢病毒，检测沉默内源性ALR后U266细胞增殖及凋亡情况。
方法：利用台盼蓝染色初步观察U266细胞株在分别加入外源性ALR和ALR单克隆抗体后的增殖情况，然后利用MTS法检测不同浓度的ALR和ALR单抗对U266细胞株增殖的影响。接下来，构建表达ALR shRNA干扰慢病毒，感染U266细胞株，在荧光显微镜下通过绿色荧光蛋白（GFP）观察感染效率，Real-time PCR检测U266细胞株ALR mRNA水平，Western blot检测U266细胞株ALR蛋白水平，观察干扰效率。通过台盼蓝排斥实验、MTS法、Brdu渗入实验检测表达ALR shRNA慢病毒干扰ALR表达对U266增殖的影响。最后，通过流式细胞计数检测U266细胞凋亡情况，Realtime PCR检测凋亡相关因子P53、Bax、Bcl-2、survivin变化情况。
结果：
1、台盼蓝染色、MTS实验结果显示外源性加入ALR能促进U266细胞的增殖，且具有一定范围内的浓度-效应关系，外源性加入抗ALR单克隆抗体能够抑制U266细胞的增殖。
2、成功构建了表达ALR shRNA干扰慢病毒，荧光显微镜显示shRNA干扰慢病毒能顺利感染U266细胞株，其MOI值为1时，感染效率达80%。
3、Real-time PCR检测表达ALR shRNA慢病毒感染U266细胞株72h后ALR mRNA水平明显降低，约为对照shRNA慢病毒组的20%左右；Western blot检测表达ALR shRNA慢病毒感染U266细胞株72h后ALR蛋白水平明显降低。
4、台盼蓝染色、MTS法、Brdu渗入实验均显示表达ALR shRNA慢病毒感染U266细胞株72h后，细胞增殖明显受抑，其差异具有统计学
意义（P<0.05）。
5、流式细胞仪检测结果显示表达ALR shRNA慢病毒感染U266细
胞株72h后细胞凋亡明显上升，Realtime PCR显示凋亡相关因子Bax明显上调，而Bcl-2明显下调，差异均具有统计学意义（P<0.05）。
结论：
1、外源性ALR能够促进人多发性骨髓瘤细胞株U266的增殖。
2、外源性中和U266细胞自分泌的ALR，能够抑制U266细胞的增
殖。
3、表达ALR shRNA干扰慢病毒能够内源性沉默ALR的表达。
4、内源性沉默ALR的表达能够抑制U266细胞的增殖，并且通过影响凋亡相关因子Bax和Bcl-2来调控U266细胞的凋亡。
第三部分：表达ALR shRNA慢病毒感染U266细胞株后多个家族细胞因子变化情况的研究。
目的：通过前两部分我们证实了ALR对U266细胞株的促增殖和抗凋亡作用，本部分利用慢病毒转染U266细胞株沉默ALR基因表达后，ELISA法检测与多发性骨髓瘤增殖和凋亡相关的多个家族细胞因子变化，筛选出可能与ALR蛋白表达相关的细胞因子，从而为进一步的机制研究奠定基础。
方法：利用ELISA法分别检测表达ALR shRNA慢病毒感染U266细胞株后细胞因子IL-6、IL-10、VEGF、TNF-α的水平。
结果：ELISA检测结果显示表达ALR shRNA慢病毒感染U266细胞株后其上清中IL-6水平明显下调，与对照组比较，其差异具有统计学意义（P<0.05）。而IL-10、VEGF、TNF-α的水平与对照组比较无统计学差异（P>0.05）。
结论：内源性沉默U266细胞株的ALR表达能够明显下调细胞因子IL-6水平，但对细胞因子IL-10、VEGF、TNF-α无影响。
Multiple myeloma（MM） is a malignant tumor derived from plasmacells. The incidence of MM is in the second place of blood systemtumor[1].Now the pathogenesis of MM is unclear.The relationship betweenmyeloma cells and cytokines in the bone marrow microvironment is ahotspot of research.A large number of studies have found interleukin-6,interleukin-10, interleukin-21,tumor necrosis factor(TNF),hepatocytegrowth factor(HGF),vascular endothelial growth factor(VEGF) and someother cytokines plays a very important role in anti-apoptosis andproliferation of myeloma[2-5],these cytokines led to myeloma cellproliferation bypass JAK/STAT3, PI-3kinase/AKT, Ras/MAPK,NF-κB,β-catenin pathways.
There’s an accidentally found that when our team product themonoclonal antibodies of augmenter of liver regeneration(ALR) using thehybridoma cells which fused by myeloma cells of BALB/c mice SP2/0andB lymphocyte of BALB/c mice,ascitic fluid with anti-ALR in two or threeof five mice vaccinated by hybridoma cells disappeared itself and there’s no any trace of hybridoma cells when we open up the abdomen andcheck.Thus we speculated that myeloma cells may produce and secrete largeamounts of ALR and strongly rely on ALR to sustain its malignantgrowth.When the ALR of myeloma neutralized by monoclonal antibodiesof ALR secreted by B lymphocyte of BALB/c mice,the ascitic fluiddisappeared for lack of stimulate signal which can maintain theproliferation of myeloma.
Augmenter of liver regeneration is a thermostable cytokines cloned inthe early1990s which can promote the repair of the liver cells. Themolecular weight of ALR is about15KD and the molecular structure andamino acid sequences are different from hepatocyte growthfactor(HGF).ALR is expressed in various tissues and organs but the researchof its biological function mainly confined to the liver[6]. ALR is highexpressed in hepatoma tissue and it can stimulate the proliferation ofhepatoma cells in dose dependent manner, but didn’t expressed in normalliver tissue and have no effect on liver primary cells[7-10].All of these suggestthat ALR played an important role in development of liver cancer.Our teamhad proved that the proliferation of hepatoma cells and the growth oftransplantation tumor can be significantly inhibited by prevent thecombination of ALR and receptor with monoclonal antibody of ALR,orblock the expression of ALR in transcription using siRNA[11、12].
There is no any research reports about the expression of ALR in MM and its effect on the proliferation of MM. Therefore,we aimed at ALR mayparticipate the development of MM,expound the mechanism,and offers anew reference target and experimental basis for the treatment of MM.
This reseach can be divided into three parts:
Part1:Study of expression of ALR in human multiple myeloma cellline U266.
Objective: To prove the high expression of ALR in multiple myelomacells by detect the mRNA level and protein level and lay the foundation forinvestigate the function of ALR in MM.
Methods: Detect the mRNA expression in human multiple myelomacell line U266, mouse myeloma cell line SP2/0and normal humanperipheral blood mononuclear cell line PMBC by Realtime PCR.Detect theprotein expression in these cell lines by Western blot.
Results: Realtime PCR results showed that the mRNAexpression ofALR in human multiple myeloma cell line U266and in mouse myelomacell line SP2/0were significantly higher than that in normal humanperipheral blood mononuclear cell line PMBC.Western blot results showedthat their were high level protein expression of ALR in U266and SP2/0,buttheir was no expression could be detected in PMBC.
Conclusion: Both on protein level and mRNA level ALR were highexpressed in human multiple myeloma cell line U266.
Part2: Study of the effection of ALR in human multiplemyeloma cell line U266proliferation and apoptosis.
Objective: Detect the effection of exogenous ALR and monoclonalantibody of ALR on the proliferation of U266.Construct lentivirus whichexpress interference gene of ALR detect proliferation and apoptosis ofU266cells after endogenous ALR gene was silenced.
Methods: Detect the proliferation of U266cells after addingexogenous ALR and monoclonal antibody of ALR by trpan blue stain andMTS. Construct lentivirus with ALR shRNA gene and observe theinfection efficiency under the fluorescence microscope by greenfluorescent protein (GFP). Detect the mRNA expression and the proteinexpression in U266cell line by Real-time PCR and western blot. Detect theU266cells proliferation infected by lentivirus with ALR shRNA gene bytrpan blue stain,MTS and BrdU incorporation. Finally, detect U266cellsapoptosis by flow cytometry and detect apoptosis related cytokinesP53,Bax, Bcl-2and survivin of U266cells by Realtime PCR.
Results:
1.Trypan blue stain and MTS experimental results showed thatexogenous ALR can promote U266cells proliferation,and this promotion has a certain range of concentration-effect relationship， exogenousmonoclonal antibody of ALR can inhibit U266cells proliferation.
2. Successfully construct lentivirus with ALR shRNA gene. Thefluorescence microscope showed when the multiplicity of infection（MOI）was1,the lentivirus can infect U266cells and the efficiency of infectionwas reached80%.
3. Realtime PCR results showed that after72h the mRNAexpression ofALR in U266cells infected by the lentivirus was significantlydecreased,it’s about1/4-1/5of the control group. Western blot resultsshowed that the protein expression of ALR in U266cells infected by thelentivirus after72h was significantly decreased too.
4. Trpan blue stain,MTS and BrdU incorporation showed that after72hthe proliferation of U266cells infected by the lentivirus was inhibited andthe difference was statistically significant(P<0.05).
5.Flow cytometry showed after72h the apoptosis of U266cellsinfected by the lentivirus increased and Realtime PCR showed thatapoptosis related cytokines Bax raised obviously, and the Bcl-2lowered,the difference was statistically significant(P<0.05).
Conclusion:
1. Exogenous ALR can promote proliferation of human multiplemyeloma cell line U266.
2. Exogenous monoclonal antibody of ALR can inhibit proliferation of human multiple myeloma cell line U266by neutralize the autocrine ALR.
3. The lentivirus with ALR shRNA gene can endogenous silence theexpression of ALR.
4. Endogenous silence the expression of ALR can inhibit proliferationof U266and regulate apoptosis of U266cells by affect apoptosis relatedfactors Bax and Bcl–2.
Part3：Study of the changes of multiple family cytokines in U266cells after infected by lentivirus with ALR shRNA gene.
Objective: We confirmed the effect on proliferation and apoptosis ofU266cell lines in the first two parts.In this part,we detected multiplefamily cytokines which associated with the proliferation and apoptosis ofU266cells infected by lentivirus. Selected cytokines which associated withALR protein expression and lay the foundation for investigate themechanism of ALR in MM.
Methods: Detect cytokines IL-6,IL-10,VEGF and TNF-αin U266cellsinfected by lentivirus by ELISA method.
Results: ELISA results showed the IL-6level decreased obviouslycompared with control group and the difference was statisticallysignificant(P<0.05). But the level of IL-10,VEGF and TNF-αshowed nostatistical difference compared with controls(P>0.05).
Conclusion: Endogenous silent ALR gene express in U266cells canobviously down-regulate cytokine IL-6level but had no effection oncytokines IL-10,VEGF and TNF-α.
我们课题组在利用BALB/c小鼠的骨髓瘤细胞SP2/0和受免疫BALB/c小鼠的B淋巴细胞融合而成的杂交瘤细胞制备抗人肝再生增强因子（Augmenter of Liver Regeneration,ALR）单抗时偶然发现：将能产生抗人ALR单抗的杂交瘤细胞注射入BALB/c小鼠腹腔内，从而产生含高浓度抗人ALR单抗的腹水，我们进行了多次制备，每次五只小鼠，然而让人惊奇的是，每次总有2-3只小鼠在产生1-2轮腹水后，腹水自行消失，开腹检查找不到任何接种杂交瘤细胞的痕迹。由此我们推测，骨髓瘤细胞可能产生和分泌大量ALR，并且严重依赖ALR来维持其恶性生长，由于小鼠的B淋巴细胞产生的抗人ALR单抗中和了骨髓瘤细胞增殖依赖的ALR，导致骨髓瘤细胞缺乏维持其恶性生长的刺激信号而发生凋亡，最终腹水自行消失。
肝再生增强因子是上世纪90年代初被克隆的一种新的热稳定性、非特异性促进损伤肝脏修复的细胞因子，分子量约为15KD，其氨基酸序列和分子结构均不同于肝细胞生长因子（HGF），它在体内多种组织器官中有表达[6]。目前对它的生物学作用研究主要局限于肝脏。我们课题组前期实验发现，ALR在体外能刺激肝癌细胞的增殖，且其促增殖效应与ALR的浓度成正相关，但是ALR对原代肝细胞的增殖无刺激作用[7、8]。实验证明ALR在肝癌组织中是高表达的，而正常成人大多数肝细胞均不表达ALR[9、10]，这些都提示ALR在肝癌的发生、发展中起了重要作用。我们课题组最新的研究（基金资助项目：30570826）也证明用ALR特异性单抗阻断肝癌细胞分泌的ALR与其受体结合或用siRNA在转录水平阻止ALR表达，均能明显抑制肝癌细胞增殖和移植瘤的生长[11、12]。
关于ALR在多发性骨髓瘤中的表达及其在多发性骨髓瘤增殖中的作用，目前国内外尚无任何研究报道。因此，我们以此为切入点，针对以上ALR是否参与多发性骨髓瘤发生、发展的作用进行研究，以期阐明ALR在多发性骨髓瘤发生、发展中的作用和机制，并为多发性骨髓瘤的治疗提供新的参考靶点和实验依据。
本研究主要分为三个部分：
第一部分：ALR在人多发性骨髓瘤细胞株U266中的表达研究。
目的：通过对人多发性骨髓瘤细胞株U266中ALR的mRNA和蛋白水平的检测，证明多发性骨髓瘤细胞高表达ALR，为进一步探讨ALR在多发性骨髓瘤细胞增殖中的作用奠定基础。
方法：利用Realtime PCR检测人多发性骨髓瘤细胞株U266、小鼠骨髓瘤细胞株SP2/0及正常人外周血单个核细胞PMBC中ALR的mRNA表达水平；利用Western-blot检测U266、SP2/0及PMBC细胞中ALR的蛋白表达水平。
结果：Realtime PCR结果显示人多发性骨髓瘤细胞株U266和小鼠骨髓瘤细胞株SP2/0中ALR的mRNA表达水平明显高于正常人外周血单个核细胞PMBC。Western-blot结果显示U266、SP2/0细胞株中ALR蛋白表达水平较高，而PMBC组未见ALR蛋白表达。
结论：在多发性骨髓瘤细胞株U266中，ALR mRNA及蛋白水平均为高表达。
第二部分：ALR对人多发性骨髓瘤细胞株U266增殖及凋亡的影响研究。
目的：检测外源性加入ALR及抗ALR单克隆抗体后U266细胞增殖情况；构建表达ALR基因的shRNA干扰慢病毒，检测沉默内源性ALR后U266细胞增殖及凋亡情况。
方法：利用台盼蓝染色初步观察U266细胞株在分别加入外源性ALR和ALR单克隆抗体后的增殖情况，然后利用MTS法检测不同浓度的ALR和ALR单抗对U266细胞株增殖的影响。接下来，构建表达ALR shRNA干扰慢病毒，感染U266细胞株，在荧光显微镜下通过绿色荧光蛋白（GFP）观察感染效率，Real-time PCR检测U266细胞株ALR mRNA水平，Western blot检测U266细胞株ALR蛋白水平，观察干扰效率。通过台盼蓝排斥实验、MTS法、Brdu渗入实验检测表达ALR shRNA慢病毒干扰ALR表达对U266增殖的影响。最后，通过流式细胞计数检测U266细胞凋亡情况，Realtime PCR检测凋亡相关因子P53、Bax、Bcl-2、survivin变化情况。
结果：
1、台盼蓝染色、MTS实验结果显示外源性加入ALR能促进U266细胞的增殖，且具有一定范围内的浓度-效应关系，外源性加入抗ALR单克隆抗体能够抑制U266细胞的增殖。
2、成功构建了表达ALR shRNA干扰慢病毒，荧光显微镜显示shRNA干扰慢病毒能顺利感染U266细胞株，其MOI值为1时，感染效率达80%。
3、Real-time PCR检测表达ALR shRNA慢病毒感染U266细胞株72h后ALR mRNA水平明显降低，约为对照shRNA慢病毒组的20%左右；Western blot检测表达ALR shRNA慢病毒感染U266细胞株72h后ALR蛋白水平明显降低。
4、台盼蓝染色、MTS法、Brdu渗入实验均显示表达ALR shRNA慢病毒感染U266细胞株72h后，细胞增殖明显受抑，其差异具有统计学
意义（P<0.05）。
5、流式细胞仪检测结果显示表达ALR shRNA慢病毒感染U266细
胞株72h后细胞凋亡明显上升，Realtime PCR显示凋亡相关因子Bax明显上调，而Bcl-2明显下调，差异均具有统计学意义（P<0.05）。
结论：
1、外源性ALR能够促进人多发性骨髓瘤细胞株U266的增殖。
2、外源性中和U266细胞自分泌的ALR，能够抑制U266细胞的增
殖。
3、表达ALR shRNA干扰慢病毒能够内源性沉默ALR的表达。
4、内源性沉默ALR的表达能够抑制U266细胞的增殖，并且通过影响凋亡相关因子Bax和Bcl-2来调控U266细胞的凋亡。
第三部分：表达ALR shRNA慢病毒感染U266细胞株后多个家族细胞因子变化情况的研究。
目的：通过前两部分我们证实了ALR对U266细胞株的促增殖和抗凋亡作用，本部分利用慢病毒转染U266细胞株沉默ALR基因表达后，ELISA法检测与多发性骨髓瘤增殖和凋亡相关的多个家族细胞因子变化，筛选出可能与ALR蛋白表达相关的细胞因子，从而为进一步的机制研究奠定基础。
方法：利用ELISA法分别检测表达ALR shRNA慢病毒感染U266细胞株后细胞因子IL-6、IL-10、VEGF、TNF-α的水平。
结果：ELISA检测结果显示表达ALR shRNA慢病毒感染U266细胞株后其上清中IL-6水平明显下调，与对照组比较，其差异具有统计学意义（P<0.05）。而IL-10、VEGF、TNF-α的水平与对照组比较无统计学差异（P>0.05）。
结论：内源性沉默U266细胞株的ALR表达能够明显下调细胞因子IL-6水平，但对细胞因子IL-10、VEGF、TNF-α无影响。
Multiple myeloma（MM） is a malignant tumor derived from plasmacells. The incidence of MM is in the second place of blood systemtumor[1].Now the pathogenesis of MM is unclear.The relationship betweenmyeloma cells and cytokines in the bone marrow microvironment is ahotspot of research.A large number of studies have found interleukin-6,interleukin-10, interleukin-21,tumor necrosis factor(TNF),hepatocytegrowth factor(HGF),vascular endothelial growth factor(VEGF) and someother cytokines plays a very important role in anti-apoptosis andproliferation of myeloma[2-5],these cytokines led to myeloma cellproliferation bypass JAK/STAT3, PI-3kinase/AKT, Ras/MAPK,NF-κB,β-catenin pathways.
There’s an accidentally found that when our team product themonoclonal antibodies of augmenter of liver regeneration(ALR) using thehybridoma cells which fused by myeloma cells of BALB/c mice SP2/0andB lymphocyte of BALB/c mice,ascitic fluid with anti-ALR in two or threeof five mice vaccinated by hybridoma cells disappeared itself and there’s no any trace of hybridoma cells when we open up the abdomen andcheck.Thus we speculated that myeloma cells may produce and secrete largeamounts of ALR and strongly rely on ALR to sustain its malignantgrowth.When the ALR of myeloma neutralized by monoclonal antibodiesof ALR secreted by B lymphocyte of BALB/c mice,the ascitic fluiddisappeared for lack of stimulate signal which can maintain theproliferation of myeloma.
Augmenter of liver regeneration is a thermostable cytokines cloned inthe early1990s which can promote the repair of the liver cells. Themolecular weight of ALR is about15KD and the molecular structure andamino acid sequences are different from hepatocyte growthfactor(HGF).ALR is expressed in various tissues and organs but the researchof its biological function mainly confined to the liver[6]. ALR is highexpressed in hepatoma tissue and it can stimulate the proliferation ofhepatoma cells in dose dependent manner, but didn’t expressed in normalliver tissue and have no effect on liver primary cells[7-10].All of these suggestthat ALR played an important role in development of liver cancer.Our teamhad proved that the proliferation of hepatoma cells and the growth oftransplantation tumor can be significantly inhibited by prevent thecombination of ALR and receptor with monoclonal antibody of ALR,orblock the expression of ALR in transcription using siRNA[11、12].
There is no any research reports about the expression of ALR in MM and its effect on the proliferation of MM. Therefore,we aimed at ALR mayparticipate the development of MM,expound the mechanism,and offers anew reference target and experimental basis for the treatment of MM.
This reseach can be divided into three parts:
Part1:Study of expression of ALR in human multiple myeloma cellline U266.
Objective: To prove the high expression of ALR in multiple myelomacells by detect the mRNA level and protein level and lay the foundation forinvestigate the function of ALR in MM.
Methods: Detect the mRNA expression in human multiple myelomacell line U266, mouse myeloma cell line SP2/0and normal humanperipheral blood mononuclear cell line PMBC by Realtime PCR.Detect theprotein expression in these cell lines by Western blot.
Results: Realtime PCR results showed that the mRNAexpression ofALR in human multiple myeloma cell line U266and in mouse myelomacell line SP2/0were significantly higher than that in normal humanperipheral blood mononuclear cell line PMBC.Western blot results showedthat their were high level protein expression of ALR in U266and SP2/0,buttheir was no expression could be detected in PMBC.
Conclusion: Both on protein level and mRNA level ALR were highexpressed in human multiple myeloma cell line U266.
Part2: Study of the effection of ALR in human multiplemyeloma cell line U266proliferation and apoptosis.
Objective: Detect the effection of exogenous ALR and monoclonalantibody of ALR on the proliferation of U266.Construct lentivirus whichexpress interference gene of ALR detect proliferation and apoptosis ofU266cells after endogenous ALR gene was silenced.
Methods: Detect the proliferation of U266cells after addingexogenous ALR and monoclonal antibody of ALR by trpan blue stain andMTS. Construct lentivirus with ALR shRNA gene and observe theinfection efficiency under the fluorescence microscope by greenfluorescent protein (GFP). Detect the mRNA expression and the proteinexpression in U266cell line by Real-time PCR and western blot. Detect theU266cells proliferation infected by lentivirus with ALR shRNA gene bytrpan blue stain,MTS and BrdU incorporation. Finally, detect U266cellsapoptosis by flow cytometry and detect apoptosis related cytokinesP53,Bax, Bcl-2and survivin of U266cells by Realtime PCR.
Results:
1.Trypan blue stain and MTS experimental results showed thatexogenous ALR can promote U266cells proliferation,and this promotion has a certain range of concentration-effect relationship， exogenousmonoclonal antibody of ALR can inhibit U266cells proliferation.
2. Successfully construct lentivirus with ALR shRNA gene. Thefluorescence microscope showed when the multiplicity of infection（MOI）was1,the lentivirus can infect U266cells and the efficiency of infectionwas reached80%.
3. Realtime PCR results showed that after72h the mRNAexpression ofALR in U266cells infected by the lentivirus was significantlydecreased,it’s about1/4-1/5of the control group. Western blot resultsshowed that the protein expression of ALR in U266cells infected by thelentivirus after72h was significantly decreased too.
4. Trpan blue stain,MTS and BrdU incorporation showed that after72hthe proliferation of U266cells infected by the lentivirus was inhibited andthe difference was statistically significant(P<0.05).
5.Flow cytometry showed after72h the apoptosis of U266cellsinfected by the lentivirus increased and Realtime PCR showed thatapoptosis related cytokines Bax raised obviously, and the Bcl-2lowered,the difference was statistically significant(P<0.05).
Conclusion:
1. Exogenous ALR can promote proliferation of human multiplemyeloma cell line U266.
2. Exogenous monoclonal antibody of ALR can inhibit proliferation of human multiple myeloma cell line U266by neutralize the autocrine ALR.
3. The lentivirus with ALR shRNA gene can endogenous silence theexpression of ALR.
4. Endogenous silence the expression of ALR can inhibit proliferationof U266and regulate apoptosis of U266cells by affect apoptosis relatedfactors Bax and Bcl–2.
Part3：Study of the changes of multiple family cytokines in U266cells after infected by lentivirus with ALR shRNA gene.
Objective: We confirmed the effect on proliferation and apoptosis ofU266cell lines in the first two parts.In this part,we detected multiplefamily cytokines which associated with the proliferation and apoptosis ofU266cells infected by lentivirus. Selected cytokines which associated withALR protein expression and lay the foundation for investigate themechanism of ALR in MM.
Methods: Detect cytokines IL-6,IL-10,VEGF and TNF-αin U266cellsinfected by lentivirus by ELISA method.
Results: ELISA results showed the IL-6level decreased obviouslycompared with control group and the difference was statisticallysignificant(P<0.05). But the level of IL-10,VEGF and TNF-αshowed nostatistical difference compared with controls(P>0.05).
Conclusion: Endogenous silent ALR gene express in U266cells canobviously down-regulate cytokine IL-6level but had no effection oncytokines IL-10,VEGF and TNF-α.
引文
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