理冲生髓饮有效组分对卵巢癌干细胞调控相关基因及顺铂耐药性的影响及其作用机制研究
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摘要
背景本课题组前期对理冲生髓饮有效组分进行了抗卵巢癌的研究,结果表明中药复方可以抑制卵巢癌细胞增殖,诱导癌细胞凋亡,减少癌细胞黏附,抑制血管新生,由此提出理冲生髓饮有效组分可以逆转卵巢癌干细胞耐药这一假说。目的探讨理冲生髓饮有效组分对卵巢癌干细胞调控相关基因及顺铂耐药性影响及其作用机制。方法2017年10月—2018年4月,选取BALB/C裸鼠48只,构建卵巢癌SKOV3荷瘤鼠模型,将裸鼠分为空白组(给予0.9%氯化钠溶液,12只)、顺铂组(给予顺铂,12只)、中药组(给予理冲生髓饮有效组分混合物,12只)、中药+顺铂组(除给予理冲生髓饮有效组分混合物外同时给予顺铂,12只)。于造模第8天开始给药,共给药18 d。最终空白组死亡4只裸鼠,顺铂组死亡3只裸鼠,中药组死亡1只裸鼠,中药+顺铂组死亡1只裸鼠。绘制各组肿瘤生长曲线。给药结束后处死裸鼠,制备肿瘤组织,HE染色观察肿瘤病理学特征,免疫荧光染色观察肿瘤CD~+_(44)、CD~+_(117)、CD_(44)、CD_(117)、CD_(133)细胞情况,PCR检测肿瘤Nanog、Oct4、ABCC1 mRNA表达水平,免疫组化法检测肿瘤Nanog、Oct4、ABCC1表达水平。结果各组在给药第1～7天肿瘤生长速度均逐渐增快;在给药第7～10天中药组和中药+顺铂组肿瘤生长速度明显变慢,而空白组和顺铂组肿瘤生长速度较快;中药组和中药+顺铂组肿瘤在第13天生长体积达到高峰,之后逐渐下降,而空白组和顺铂组肿瘤生长速度仍逐渐增快。顺铂组肿瘤细胞数目明显减少,体积缩小,核分裂象可见,可见大量肿瘤细胞凋亡,间质纤维组织明显增生;中药组肿瘤细胞数目减少,肿瘤组织呈较明显的片状坏死,间质中纤维组织和纤维细胞增生;中药+顺铂组肿瘤细胞数目明显减少,可见大量肿瘤细胞凋亡,间质纤维组织明显增生。肿瘤中可见少量CD~+_(44)、CD~+_(117)、CD_(133)、CD_(44)、CD_(117)细胞。顺铂组、中药组、中药+顺铂组肿瘤Nanog、Oct4 m RNA表达水平高于空白组(P<0.05);中药组肿瘤Nanog mRNA表达水平高于顺铂组、中药+顺铂组(P<0.05);中药+顺铂组肿瘤ABCC1 mRNA表达水平低于空白组、顺铂组、中药组(P<0.05)。顺铂组、中药+顺铂组肿瘤Nanog表达水平低于空白组(P<0.05);中药组肿瘤Oct4表达水平低于空白组、顺铂组(P<0.05);中药组、中药+顺铂组肿瘤ABCC1表达水平低于空白组、顺铂组(P<0.05)。结论理冲生髓饮有效组分可通过下调ABCC1及其m RNA表达水平,增高肿瘤细胞中药物浓度,还可通过影响卵巢癌干细胞特性和顺铂敏感性,从而逆转卵巢癌对顺铂的耐药性。
Background The first stage of this research is focused on the anti-tumor in human ovary based on the active component of Lichong Shengsuiyin.According to the result of the survey,the Chinese herbal compound is effective in inhibiting cell proliferation of ovarian cancer,contributing to the decrease of cancer cells,alleviating their adhesion and restraining angiogenesis.On this basis,the hypothesis of whether active components of Lichong Shengmuiyin can reverse the drug resistance of ovarian cancer stem cells is put forward.Objective To investigate the effect of active components of Lichong Shengsuiyin on regulating genes of ovarian cancer stem cell and cisplatin resistance and analyze its mechanism.Methods A tumor-bearing mouse model was gradually set up using 48 nude mice of BALB/C between October 2017 and April 2018 which were divided into four groups——12 in the blank group(given 0.9% of sodium chloride solution);12 in the cisplatin group(given cisplatin);12 in the herbal group(given mixed active component of Lichong Shengsuiyin);and 12 in the herb-cisplatin group(given the active component of Lichong Shengsuiyin combined with cisplatin).On day 8 after the establishment of the model,drug was used for 18 d.In the end,4 nude mice in the blank group died,3 in the cisplatin group died,1 in the herbal group died and 1 in the herb-cisplatin group died.At the same time,the growth curve of the tumor in all groups were drawn.Once the drug use was over,the mice were killed,and the pathological feature of the tumors was observed by HE staining.CD_(44)~+,CD_(117)~+,CD_(44),CD_(117) and CD_(133) of tumor cells was also observed by immunofluorescent staining.The expression levels of tumor Nanog,Oct4,and ABCC1 mRNA were tested by Polymerase Chain Reaction(PCR).Nanog,Oct4 and ABCC1 expression levels in tumors were detected by immunohistochemistry.Results In the first 7 days after the drug use,the growth speed of tumor of all group increased gradually.In the next three days,however,the growth speed of tumor in herbal group and herb-cisplatin group decreased noticeably,while continued fast growth occurred in the blank group and cisplatin group.And the 13 th day witnessed the maximum increase of the growth volume of tumors in herbal group and herb-cisplatin group,which was later followed by a gradual decrease,while that in the blank group and cisplatin group continued to grow.In the cisplatin group,the number of tumor cells reduced obviously,the volume shrank,the mitotic figure was noticed,a large number of cells died and interstitial fiber proliferated.In the herbal group,the tumor cells reduced,patchy necrosis was common among tumor tissues,while fiber tissues and cells proliferated.In the herb-cisplatin group,the number of tumor cells decreased dramatically with large quantities of cells dying and obvious proliferation among fiber tissues.Besides,a few CD_(44)~+,CD_(117)~+,CD_(133),CD_(44) and CD_(117) cells were noticeable among tumors.The expression level of tumor Nanog and Oct4 mRNA was higher in the cisplatin group,herbal group and herb-cisplatin group than that in the blank group(P<0.05);expression level of tumor Nanog mRNA was higher in the herbal group than that in the cisplatin group and herb-cisplatin group(P<0.05);expression level of tumor ABCC1 mRNA in the herb-cisplatin group was lower than that in others(P<0.05);expression level of tumor Nanog in the cisplatin group and herb-cisplatin group was lower than that in the blank group(P<0.05);expression level of tumor Oct4 was higher in the herbal group than that in the blank group and cisplatin group(P<0.05);expression level of tumor ABCC1 was higher in the blank group and the cisplatin group than that in the herbal group and herb-cisplatin group(P<0.05).Conclusion The active component of Lichong Shengsuiyin,once deduced in the expression level of ABCC1 and its mRNA,can slow down the flow of drug,hence raising its concentration in tumor cells.In addition,it can help to reverse the cisplatin drug resistance of ovarian cancer by affecting the traits of its stem cells and the sensitivity of cisplatin.
Background The first stage of this research is focused on the anti-tumor in human ovary based on the active component of Lichong Shengsuiyin.According to the result of the survey,the Chinese herbal compound is effective in inhibiting cell proliferation of ovarian cancer,contributing to the decrease of cancer cells,alleviating their adhesion and restraining angiogenesis.On this basis,the hypothesis of whether active components of Lichong Shengmuiyin can reverse the drug resistance of ovarian cancer stem cells is put forward.Objective To investigate the effect of active components of Lichong Shengsuiyin on regulating genes of ovarian cancer stem cell and cisplatin resistance and analyze its mechanism.Methods A tumor-bearing mouse model was gradually set up using 48 nude mice of BALB/C between October 2017 and April 2018 which were divided into four groups——12 in the blank group(given 0.9% of sodium chloride solution);12 in the cisplatin group(given cisplatin);12 in the herbal group(given mixed active component of Lichong Shengsuiyin);and 12 in the herb-cisplatin group(given the active component of Lichong Shengsuiyin combined with cisplatin).On day 8 after the establishment of the model,drug was used for 18 d.In the end,4 nude mice in the blank group died,3 in the cisplatin group died,1 in the herbal group died and 1 in the herb-cisplatin group died.At the same time,the growth curve of the tumor in all groups were drawn.Once the drug use was over,the mice were killed,and the pathological feature of the tumors was observed by HE staining.CD_(44)~+,CD_(117)~+,CD_(44),CD_(117) and CD_(133) of tumor cells was also observed by immunofluorescent staining.The expression levels of tumor Nanog,Oct4,and ABCC1 mRNA were tested by Polymerase Chain Reaction(PCR).Nanog,Oct4 and ABCC1 expression levels in tumors were detected by immunohistochemistry.Results In the first 7 days after the drug use,the growth speed of tumor of all group increased gradually.In the next three days,however,the growth speed of tumor in herbal group and herb-cisplatin group decreased noticeably,while continued fast growth occurred in the blank group and cisplatin group.And the 13 th day witnessed the maximum increase of the growth volume of tumors in herbal group and herb-cisplatin group,which was later followed by a gradual decrease,while that in the blank group and cisplatin group continued to grow.In the cisplatin group,the number of tumor cells reduced obviously,the volume shrank,the mitotic figure was noticed,a large number of cells died and interstitial fiber proliferated.In the herbal group,the tumor cells reduced,patchy necrosis was common among tumor tissues,while fiber tissues and cells proliferated.In the herb-cisplatin group,the number of tumor cells decreased dramatically with large quantities of cells dying and obvious proliferation among fiber tissues.Besides,a few CD_(44)~+,CD_(117)~+,CD_(133),CD_(44) and CD_(117) cells were noticeable among tumors.The expression level of tumor Nanog and Oct4 mRNA was higher in the cisplatin group,herbal group and herb-cisplatin group than that in the blank group(P<0.05);expression level of tumor Nanog mRNA was higher in the herbal group than that in the cisplatin group and herb-cisplatin group(P<0.05);expression level of tumor ABCC1 mRNA in the herb-cisplatin group was lower than that in others(P<0.05);expression level of tumor Nanog in the cisplatin group and herb-cisplatin group was lower than that in the blank group(P<0.05);expression level of tumor Oct4 was higher in the herbal group than that in the blank group and cisplatin group(P<0.05);expression level of tumor ABCC1 was higher in the blank group and the cisplatin group than that in the herbal group and herb-cisplatin group(P<0.05).Conclusion The active component of Lichong Shengsuiyin,once deduced in the expression level of ABCC1 and its mRNA,can slow down the flow of drug,hence raising its concentration in tumor cells.In addition,it can help to reverse the cisplatin drug resistance of ovarian cancer by affecting the traits of its stem cells and the sensitivity of cisplatin.
引文
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[6]FFRENCH B,GASCH C,O'LEARY J J,et al.Developing ovarian cancer stem cell models:laying the pipeline from discovery to clinical intervention[J].Mol Cancer,2014,13:262.DOI:10.1186/1476-4598-13-262.
[7]CORBEIL D,R?PER K,HELLWIG A,et al.The human AC133hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions[J].J Biol Chem,2000,275(8):5512-5520.
[8]CURLEY M D,THERRIEN V A,CUMMINGS C L,et al.CD133expression defines a tumor initiating cell population in primary human ovarian cancer[J].Stem Cells,2009,27(12):2875-2883.DOI:10.1002/stem.236.
[9]STEWART J M,SHAW P A,GEDYE C,et al.Phenotypic heterogeneity and instability of human ovarian tumor-initiating cells[J].Proc Natl Acad Sci U S A,2011,108(16):6468-6473.DOI:10.1073/pnas.1005529108.
[10]KUSUMBE A P,MALI A M,BAPAT S A.CD133-expressing stem cells associated with ovarian metastases establish an endothelial hierarchy and contribute to tumor vasculature[J].Stem Cells,2009,27(3):498-508.DOI:10.1634/stemcells.2008-0868.
[11]SKUBITZ A P,TARAS E P,BOYLAN K L,et al.Targeting CD133 in an in vivo ovarian cancer model reduces ovarian cancer progression[J].Gynecol Oncol,2013,130(3):579-587.DOI:10.1016/j.ygyno.2013.05.027.
[12]CHAMBERS I,COLBY D,ROBERTSON M,et al.Functional expression cloning of Nanog,a pluripotency sustaining factor in embryonic stem cells[J].Cell,2003,113(5):643-655.
[13]MITSUI K,TOKUZAWA Y,ITOH H,et al.The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J].Cell,2003,113(5):631-642.
[14]CAVALERI F,SCH?LER H R.Nanog:a new recruit to the embryonic stem cell orchestra[J].Cell,2003,113(5):551-552.
[15]HOLLAND M L,ALLEN J D,ARNOLD J C.Interaction of plant cannabinoids with the multidrug transporter ABCC1(MRP1)[J].EurJPharmacol,2008,591(1/3):128-131.DOI:10.1016/j.ejphar.2008.06.079.
[2]EISENHAUER E A,VERMORKEN J B,VAN GLABBEKE M.Predictors of response to subsequent chemotherapy in platinum pretreated ovarian cancer:a multivariate analysis of 704 patients[seecomments][J].Ann Oncol,1997,8(10):963-968.
[3]MEINHOLD-HEERLEIN I,HAUPTMANN S.The heterogeneity of ovarian cancer[J].Arch Gynecol Obstet,2014,289(2):237-239.DOI:10.1007/s00404-013-3114-3.
[4]韩凤娟,隋丽华,马荣,等.理冲生髓饮对卵巢上皮性癌患者免疫状态影响的临床研究[J].中医药信息,2003,20(2):37-38.DOI:10.3969/j.issn.1002-2406.2003.02.018.
[5]ZHANG S,BALCH C,CHAN M W,et al.Identification and characterization of ovarian cancer-initiating cells from primary human tumors[J].Cancer Res,2008,68(11):4311-4320.DOI:10.1158/0008-5472.CAN-08-0364.
[6]FFRENCH B,GASCH C,O'LEARY J J,et al.Developing ovarian cancer stem cell models:laying the pipeline from discovery to clinical intervention[J].Mol Cancer,2014,13:262.DOI:10.1186/1476-4598-13-262.
[7]CORBEIL D,R?PER K,HELLWIG A,et al.The human AC133hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions[J].J Biol Chem,2000,275(8):5512-5520.
[8]CURLEY M D,THERRIEN V A,CUMMINGS C L,et al.CD133expression defines a tumor initiating cell population in primary human ovarian cancer[J].Stem Cells,2009,27(12):2875-2883.DOI:10.1002/stem.236.
[9]STEWART J M,SHAW P A,GEDYE C,et al.Phenotypic heterogeneity and instability of human ovarian tumor-initiating cells[J].Proc Natl Acad Sci U S A,2011,108(16):6468-6473.DOI:10.1073/pnas.1005529108.
[10]KUSUMBE A P,MALI A M,BAPAT S A.CD133-expressing stem cells associated with ovarian metastases establish an endothelial hierarchy and contribute to tumor vasculature[J].Stem Cells,2009,27(3):498-508.DOI:10.1634/stemcells.2008-0868.
[11]SKUBITZ A P,TARAS E P,BOYLAN K L,et al.Targeting CD133 in an in vivo ovarian cancer model reduces ovarian cancer progression[J].Gynecol Oncol,2013,130(3):579-587.DOI:10.1016/j.ygyno.2013.05.027.
[12]CHAMBERS I,COLBY D,ROBERTSON M,et al.Functional expression cloning of Nanog,a pluripotency sustaining factor in embryonic stem cells[J].Cell,2003,113(5):643-655.
[13]MITSUI K,TOKUZAWA Y,ITOH H,et al.The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J].Cell,2003,113(5):631-642.
[14]CAVALERI F,SCH?LER H R.Nanog:a new recruit to the embryonic stem cell orchestra[J].Cell,2003,113(5):551-552.
[15]HOLLAND M L,ALLEN J D,ARNOLD J C.Interaction of plant cannabinoids with the multidrug transporter ABCC1(MRP1)[J].EurJPharmacol,2008,591(1/3):128-131.DOI:10.1016/j.ejphar.2008.06.079.