人锌指蛋白23在原发性肝细胞肝癌中的表达及临床意义

目的 分析hZNF23在人HCC组织及HepG2细胞系中的表达情况,以探讨其与HCC临床病理特征和细胞凋亡的关系.方法 采用实时荧光定量RT-PCR法检测37例HCC患者(按Edmondson分为两组,Ⅰ+Ⅱ级17例,Ⅲ+Ⅳ级20例;按临床分为3组,HCC合并肝硬化和HBV感染组31例,HCC合并肝硬化和HCC合并HRV感染组各3例)癌组织和其癌旁肝组织标本中hZNF23及内参GAPDH mRNA的表达水平,并分析其与HCC临床病理特征的关系;体外培养HepG2细胞后,分为4组(对照组、1.25、2.5和5 μg/ml顺铂组)或6组(对照组、1.25、2.5、5、10和20μg/ml顺铂组).采用四唑盐比色法(MTT)和流式细胞仪膜联蛋白V/碘化丙啶染色法检测与观察HepG2细胞的增殖及凋亡情况;实时荧光定量RT-PCR法检测HepG2细胞在不同浓度的顺铂诱导凋亡后,hZNF23及内参GAPDH mRNA的表达水平.结果 37例HCC患者癌组织及其癌旁组织标本中hZNF23相对表达量分别为8.84(3.59～15.05)和22.20(13.85～42.90),差异有统计学意义(U=259.5,P＜0.01).Edmondson Ⅰ+Ⅱ级HCC组织hZNF23 mRNA相对表达量为12.80(4.80～19.50),高于Ⅲ+Ⅳ级的5.01(2.88～11.68),且差异有统计学意义(U=99.00,P＜0.05);合并肝硬化组为9.92(3.80～15.25),未合并肝硬化组为3.21(2.78～3.60)、合并HBV感染组为9.09(3.72～15.25),无HBV感染组为2.48(1.79～12.10),合并肝硬化组与未合并肝硬化组、合并HBV感染组与未合并HBV感染组比较,其hZNF23 mRNA表达量差异均无统计学意义(U值分别为16.00和24.00,P均＞0.05).顺铂作用于HepG2细胞24 h后,用MTT检测,顺铂明显抑制HepG2细胞增殖;膜联蛋白V/碘化丙啶染色法检测细胞凋亡率依次为(0.9±0.2)%、(4.2±0.3)%、(9.8±4.3)%、(23.0±6.0)%,顺铂显著诱导HepG2细胞凋亡,且呈剂量依赖性(F=27.890,P＜0.01).实时荧光定量RT-PCR法检测1.25、2.5、5μg顺铂诱导的HepGR细胞组hZNF23 mRNA相对表达量依次为(10.39±3.08)×10-5、(24.10±2.09)×10-5、(6.90±2.24)×10-4,均显著高于对照组[(9.48±1.80)×10-5,F=6.027,P＜0.01].结论 HCC组织中hZNF23相对表达水平与Edmondson分级密切关联;顺铂对HepG2细胞的凋亡作用可能与hZNF23上调有关.

Abstract：

Objective To detect the expression level of human zinc finger 23 (ZNF23) in hepatocellular carcinoma tissue samples and HepG2 cell lines and investigate the relationship between hZNF23 expression and clinicopathological characteristics of HCC and cell apoptosis. Methods The expression levels of hZNF23 and GAPDH mRNA in 37 cases of HCC were measured by real-time RT-PCR. The association between the expression of hZNF23 and the clinicopathological characteristics of HCC was analyzed. Cultured HepG2 cells were divided into 4 groups ( control group, 1.25 μg/ml , 2.5 μg/ml and 5 μg/ml cisplatin)or 6 groups( control group, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml cisplatin). MTT method was employed to evaluate cell proliferation. Annexin V-FITC assay was used to assess percentage of apoptotic HepG2 cells. The expression levels of hZNF23 and GAPDH mRNA of HepG2 cells after apoptosis induced by cisplatin with a series of concentrations were measured by real-time RT-PCR.Results The median ( quartile1, quartile 3) expression levels of hZNF23 mRNA in 37 HCC tissue samples and adjacent tissue samples were 8.84 (3.59-15.05), 22.20 ( 13.85-42.90 ), respectively. There was significant difference ( U = 259.5, P ＜ 0.01 ). The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in cancer tissue samples with Edmondson stage Ⅰ + Ⅱ [12.80(4.80-19.50)] was much higher than those in stage Ⅲ + Ⅳ [5.01 ( 2.88-11.68 ), U = 99.00, P ＜ 0.05] The median ( quartile1,quartile 3 ) expression levels of hZNF23 mRNA in patients with and without hepatic cirrhosis were 9.92(3.80-15.25) , 3.21 (2.78-3.60), respectively. The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in HBV infection and non-infection patients was 9.09(3.72-15.25 ), 2.48 (1.79-12.10),respectively. There was no significant difference between groups with and without hepatic cirrhosis and between HBV infection and non-infection groups( U = 16. 00 and 24.00, P ＞0.05 ). MTT assay indicated that cisplatin significantly inhibited HepG2 cells proliferation in a dose-dependent manner. Annexin V-FITC/PI assay showed that HepG2 cells apoptosis rates were (0.9 ± 0.2 ) %, ( 4. 2 ± 0.3 ) %, ( 9.8 ± 4. 3 ) %,(23.0 ± 6.0)%, respectively. Cisplatin significantly induced HepG2 cells apoptosis in a dose-dependent manner( F = 27.89, P ＜ 0.01 ). The expression levels of hZNF23 mRNA in cisplatin groups [( 10.39 ±3.08) × 10-5, (24.10 ± 2.09) × 10-5, (6.90 ± 2.24) × 10-4] were significantly lower than that of the controlgroup[(94.80±1.80) ×10-5, F=6.027, P＜0.01]. Conclusions The expression level hZNF23 mRNA is related to Edmondson stage of HCC. The apoptosis effect of cisplatin on HepG2 cells may be associated with the upregulation of hZNF23.     

人锌指蛋白23在原发性肝细胞肝癌中的表达及临床意义

Objective To detect the expression level of human zinc finger 23 (ZNF23) in hepatocellular carcinoma tissue samples and HepG2 cell lines and investigate the relationship between hZNF23 expression and clinicopathological characteristics of HCC and cell apoptosis. Methods The expression levels of hZNF23 and GAPDH mRNA in 37 cases of HCC were measured by real-time RT-PCR. The association between the expression of hZNF23 and the clinicopathological characteristics of HCC was analyzed. Cultured HepG2 cells were divided into 4 groups ( control group, 1.25 μg/ml , 2.5 μg/ml and 5 μg/ml cisplatin)or 6 groups( control group, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml cisplatin). MTT method was employed to evaluate cell proliferation. Annexin V-FITC assay was used to assess percentage of apoptotic HepG2 cells. The expression levels of hZNF23 and GAPDH mRNA of HepG2 cells after apoptosis induced by cisplatin with a series of concentrations were measured by real-time RT-PCR.Results The median ( quartile1, quartile 3) expression levels of hZNF23 mRNA in 37 HCC tissue samples and adjacent tissue samples were 8.84 (3.59-15.05), 22.20 ( 13.85-42.90 ), respectively. There was significant difference ( U = 259.5, P ＜ 0.01 ). The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in cancer tissue samples with Edmondson stage Ⅰ + Ⅱ [12.80(4.80-19.50)] was much higher than those in stage Ⅲ + Ⅳ [5.01 ( 2.88-11.68 ), U = 99.00, P ＜ 0.05] The median ( quartile1,quartile 3 ) expression levels of hZNF23 mRNA in patients with and without hepatic cirrhosis were 9.92(3.80-15.25) , 3.21 (2.78-3.60), respectively. The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in HBV infection and non-infection patients was 9.09(3.72-15.25 ), 2.48 (1.79-12.10),respectively. There was no significant difference between groups with and without hepatic cirrhosis and between HBV infection and non-infection groups( U = 16. 00 and 24.00, P ＞0.05 ). MTT assay indicated that cisplatin significantly inhibited HepG2 cells proliferation in a dose-dependent manner. Annexin V-FITC/PI assay showed that HepG2 cells apoptosis rates were (0.9 ± 0.2 ) %, ( 4. 2 ± 0.3 ) %, ( 9.8 ± 4. 3 ) %,(23.0 ± 6.0)%, respectively. Cisplatin significantly induced HepG2 cells apoptosis in a dose-dependent manner( F = 27.89, P ＜ 0.01 ). The expression levels of hZNF23 mRNA in cisplatin groups [( 10.39 ±3.08) × 10-5, (24.10 ± 2.09) × 10-5, (6.90 ± 2.24) × 10-4] were significantly lower than that of the controlgroup[(94.80±1.80) ×10-5, F=6.027, P＜0.01]. Conclusions The expression level hZNF23 mRNA is related to Edmondson stage of HCC. The apoptosis effect of cisplatin on HepG2 cells may be associated with the upregulation of hZNF23.     

RPA1在肝癌组织中的表达及临床意义的研究

目的观察复制蛋白A1(RPA1)在肝细胞肝癌组织中的表达并探讨其临床意义。方法应用逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)检测30例肝细胞肝癌组织及癌旁组织中RPA1 mRNA及蛋白的半定量表达水平,结合临床病理资料,统计分析肝细胞肝癌患者中RPA1表达的临床意义。结果 RPA1 mRNA在肝细胞肝癌组织的表达高于癌旁组织(0.635±0.228 vs.0.448±0.186,P0.05)。RPA1蛋白在肝细胞肝癌组织的表达高于癌旁组织(0.876±0.205 vs 0.727±0.153,P0.05)。在肝细胞肝癌组织中,RPA1 mRNA的表达在临床分期Ⅰ+Ⅱ组低于Ⅲ+Ⅳ组,组间差异有统计学意义(0.471±0.190 vs.0.744±0.184,P0.05)。在肝细胞肝癌组织中,RPA1蛋白的表达在临床分期为Ⅰ+Ⅱ组低于Ⅲ+Ⅳ组,组间差异有统计学意义(0.742±0.155 vs.0.965±0.188,P0.05)。在肝细胞肝癌组织中,RPA1 mRNA和蛋白的表达水平在患者不同年龄、性别、肝癌组织分级、是否有乙肝表面抗原、淋巴转移、门脉癌栓等临床病理特征中差异无统计学意义(P0.05)。结论 RPA1在肝细胞肝癌组织中表达上调,其表达上调可能促进肝癌的发生和发展。     

肝细胞肝癌中PED/PEA-15 mRNA及蛋白表达的临床意义

目的:探讨PED/PEA-15 mRNA及蛋白在肝癌组织、癌旁组织及正常肝组织中的表达情况及其临床意义.方法:应用半定量逆转录.聚合酶链反应检测40例肝细胞肝癌、相应癌旁组织和12例正常肝组织中PED/PEA.15基因的相对表达量.并通过免疫组化检测PED/PEA-15蛋白的表达.结果:半定量RT-PCR显示,PED/PEA-15基因在肝癌组织中均呈高表达,癌旁组织及正常肝组织呈低表达或不表达,相对表达量分别为1.201±0.301、0.64±0.101和0.565±0.077,PED/PEA-15 mRNA在肝癌组织中表达较癌旁组织、正常肝组织显著升高(P<0.01).免疫组织化学显示,肝癌组织、癌旁组织及正常肝组织中PED/PEA-15蛋白的表达率分别为72.5%、32.5%和16.7%.肝癌组织中PED/PEA-15蛋白表达较癌旁组织及正常肝组织显著升高(P<0.01).且PED/PEA-15 mRNA及蛋白的表达与肝癌病理分级、临床分期有关(P<0.05),而与发病年龄、性别、肿瘤大小、肿瘤数目差异无显著性(P>0.05).结论:PED/PEA-15 mRNA及蛋白在肝癌组织中表达水平明显升高,可能在肝癌的发生及发展过程中发挥重要作用.     

环氧化酶-2在肝细胞癌中的表达及临床意义

目的 观察环氧化酶-2(COX-2)在肝细胞癌(HCC,简称肝癌)中的表达特征.方法 应用免疫组织化学S-P法、逆转录聚合酶链反应检测重庆医科大学附属第二医院2005年10月至2007年6月手术切除并经病理切片证实的52例HCC标本、52例癌旁组织及10例正常肝组织中COX-2及其基因的表达情况.结果 COX-2/COX-2 mRNA在肝癌组织中的表达明显上调,与其在正常肝组织、癌旁组织中的表达相比差异有统计学意义(P<0.01).结论 (1)COX-2高表达参与了肝癌的发生和发展,其表达水平与肝癌的分化程度及是否合并乙型肝炎及肝硬化有关,与HCC组织的国际癌症病期分期、肿瘤大小、甲胎蛋白水平、大体类型及是否转移等无明显关系.(2)COX-2抑制剂有望为预防HCC的发生和发展提供一条新的途径.     

RNA结合蛋白Sam68 mRNA在原发性肝癌中的表达及意义

目的探讨RNA结合蛋白Sam68 mRNA在人原发性肝癌组织中的表达及意义。方法选取20例原发性肝癌的肿瘤组织及相应癌旁组织,采用TRIzol一步法提取总RNA,采用RT-PCR法检测Sam68 mRNA在肝癌组织及相应癌旁组织中的表达。结果PCR产物电泳显示肝癌组织中Sam68 mRNA平均表达强度为60.71%±9.26%,相应癌旁组织中Sam68 mRNA平均表达强度为76.98%±11.61%,经配对t检验,P〈0.05,具有统计学差异。结论与相应癌旁组织相比,人原发性肝癌组织中Sam68 mRNA的表达明显降低,提示在人原发性肝癌组织中Sam68的表达在转录水平受到明显抑制。     

结直肠癌组织中锌指蛋白139(ZNF139)的表达及临床意义研究

目的 探究锌指蛋白139(zinc finger protein 139,ZNF139)在结直肠癌(colorectal cancer)组织中的表达及与临床病理参数及预后的关系。方法 选取2012年5月～2014年6月就诊于恩施土家族苗族自治州中心医院行手术治疗的67例结直肠癌患者的临床病理资料及癌组织、癌旁组织和正常黏膜组织进行研究。采用实时荧光定量逆转录聚合酶链反应(RT-PCR)和免疫组织化学法染色检测ZNF139 mRNA及蛋白在结直肠癌组织中的表达情况,分析其与结直肠癌患者临床病理因素的关系;采用K-M生存曲线分析ZNF139表达对预后的影响;采用COX回归模型分析影响结直肠癌患者预后的风险因素。结果 结直肠癌病灶组织中ZNF139 mRNA(0.651±0.130)表达水平明显高于癌旁组织(0.217±0.070)和正常组织(0.201±0.052),差异有统计学意义(t=24.060, 24.947,均P=0.001)。结直肠癌组织中ZNF139蛋白阳性率(52.24%)明显高于癌旁组织(28.36%)和正常黏膜组织(19.40%),差异均有统计学意义(χ²     

Numb与β-catenin在肝细胞肝癌组织中的表达及临床意义

目的探讨Numb蛋白和β—catenin蛋白在肝细胞肝癌（HCC）组织中的表达及与临床病理特征的关系。方法应用免疫组检测40例HCC患者的组织、癌旁组织和40例非肝病患者的正常肝组织中Numb、β-catenin的表达,并对Numb、β—catenin的表达情况及与HCC的临床病理特征进行分析。结果Numb在正常肝组织、癌旁组织中表达呈强阳性,在HCC组织中表达呈弱阳性;β—catenin在正常肝组织、癌旁组织中表达正常,在HCC组织中表达阳性。Numb、β—catenin的表达水平与HCC患者性别、年龄及肿瘤大小无关（P〉0．05）,与HCC临床分期、门脉癌栓有关（P〈0．05）;HCC组织中Numb、β—catenin的异常表达呈负相关（r=-0．587,P〈0．01）。结论Num、β—catenin在HCC组织中的异常表达,提示二者可能参与肝癌的发生发展过程。Numb、β—catenin的异常表达与HCC门脉癌栓、临床分期有关,提示联合检测Numb、β—cateniu可作为HCC早期诊断,并对判断转移及临床分期有重要意义。     

胰岛素样生长因子结合蛋白-5在肝硬化、原发性肝癌患者肝组织和血清中的水平及临床意义

目的观察肝硬化、原发性肝癌患者肝组织和血清中胰岛素样生长因子结合蛋白-5(IGFBP-5)水平,探讨其与肝硬化、原发性肝癌的关系及其临床意义。方法取肝硬化组织、肝癌组织及癌旁正常组织各8例,采用实时RT-PCR技术检测IGFBP-5mRNA的表达水平;取肝硬化(21例)、原发性肝癌(14例)及正常对照者(14例)血清,采用ELISA法检测IGFBP-5水平,分析IGFBP-5在不同分组中水平的差异及其临床意义。结果肝硬化组肝组织中IG-FBP-5mRNA的表达及血清中IGFBP-5水平均较正常对照组降低(P均<0.05),与血清中升高的透明质酸(HA)、层粘连蛋白(LN)和肝功能无相关性。原发性肝癌组肝组织中IGFBP-5的表达及血清中IGFBP-5水平均高于正常对照组和肝硬化组(P均<0.05),与血清中升高的甲胎蛋白(AFP)和肝功能亦无相关性。结论在肝硬化发生后,肝组织表达和合成IGFBP-5减少,与其他细胞因子的调节有关,其可能与HA、LN互补作为诊断早期肝纤维化的一个无创指标;原发性肝癌患者肝组织中IGFBP-5表达增多,血清水平增高,其有可能作为一个潜在的诊断原发性肝癌的检测指标。IGFBP-5与肝硬化、原发性肝癌的关系值得进一步研究。     

凋亡相关蛋白bax在肝细胞癌的表达及其意义

目的：探讨原发性肝细胞癌（简称肝癌）细胞凋亡相关蛋白bax的表达情况及其意义。方法：采用免疫组化ABC法检测bax蛋白在66例肝癌组织和24例正常肝组织,以及3种人肝癌细胞系HCC－9204、SMMC－7721、HHCC和1种人正常肝细胞系QZG的表达情况,并分析肝癌组织的bax蛋白表达阳性率与其病理分级间的关系。结果：bax蛋白在正常肝组织中表达阳性率为70．8％（17／24）,在肝癌组织中表达阳性率为43．9％（29／66）,二者差异显著（P＜0．05）。肝癌组织的bax蛋白表达阳性率与其病理分级间未见明显相关性（P＞0．05）。肝细胞系HCC－9204、SMMC－7721、HHCC的阳性细胞计数率分别为43．2％、58．3％和47．1％。正常肝细胞系QZG的阳性细胞计数率为61．0％。结论：与正常肝组织相比,肝癌组织可出现bax蛋白表达下降。肝癌细胞系和正常肝细胞系均可见中等程度的bax蛋白表达。     

人神经元特异性烯醇化酶在肿瘤细胞系中的表达检测及意义

目的 探讨从蛋白及细胞水平检测人神经元特异性烯醇化酶（NSE）在非神经内分泌性肿瘤细胞系中的表达及其意义。方法 用NSE特异引物和逆转录-聚合酶链反应（RT-PCR）、蛋白印迹、免疫细胞化学（ICC）染色技术检测NSE在肿瘤细胞中的表达。结果 从转录蛋白及细胞水平都能从黑色素瘤细胞株WM451和WM983A、前列腺癌高转移细胞株PC-3M和低转移细胞株PC-3、乳腺癌细胞株MCF-7、结直肠癌细胞株HT-29和SL180、胃癌细胞株MGC-803中检测到NSE的表达，阳性率为100％。结论 NSE除在神经内分泌细胞表达外，在肿瘤细胞亦有更为广泛的表达谱，其作为神经内分泌来源肿瘤标志物的作用值得深入研究。     

胎盘生长因子在肝癌组织中的表达及意义

目的:研究胎盘生长因子(PLGF)在肝癌组织中的表达,探讨其与肝癌的联系.方法:收集标本:肝细胞肝癌组织49例及正常肝组织8例,用免疫组化方法和逆转录聚合酶链式反应 (RT-PCR)技术检测PLGF的表达.结果:PLGF蛋白和mRNA在肝癌组织有高水平表达,与正常肝组织比较差异有显著性意义(P ＜ 0.01).结论:PLGF与肝细胞肝癌的发生有一定的关联,可能在肝肿瘤血管新生过程中起重要作用.     

鼻咽癌患者外周血中CK20 mRNA的表达及其临床意义

目的研究鼻咽癌(NPC)患者外周血中CK20mRNA表达并探讨其临床意义。方法采用巢式逆转录和多聚酶链反应方法,对NPC伴颈淋巴结转移组19例和NPC颈部淋巴结无转移组13例(共计32例)CK20mRNA表达进行检测。CT证实两组患者均无远处转移病灶。对照组1为8例非肿瘤患者外周血;阳性对照分别为B95-8细胞株和NPC组织。结果NPC伴颈淋巴结转移组CK20mRNA阳性表达率为21.1%(4/19),颈部淋巴结无转移组为7.7%(1/13),两组间阳性表达率比较差异无显著性意义(P>0.05)。CK20mRNA阳性表达与鼻咽癌国际抗癌联盟分期无关。对照组1均为阴性表达。结论NPC患者外周血中CK20mRNA的阳性表达作为NPC细胞血行播散的指标可能是有价值的,有助于评估NPC患者的预后。     

The tumor-suppressor gene LZTS1 suppresses hepatocellular carcinoma proliferation by impairing PI3K/Akt pathway

BackgroundTo study the role of LZTS1 in hepatocellular carcinoma (HCC) proliferation and the molecular mechanism involved.MethodsLZTS1 expression was studied in 10 HCC cell lines and 1 normal hepatocyte cell line by western blot analysis and qRT-PCR. One HCC cell line was selected and transfected with LZTS1 lentivirus. Cell proliferation and cell cycle were then determined by CCK-8 assay and flow cytometry, respectively. LZTS1, cyclin D1, CDK1, Cdc25C, pS473 Akt, and pT308 Akt mRNA and protein expressions were measured. PS473 Akt and pT308 Akt expression level was also compared with the HCC cells treated with LY294002.ResultsCompared with the normal hepatocyte cells, LZTS1 expression in HCC cells was significantly lower. After the transfection with LZTS1 lentivirus, HCC cell proliferation ability decreased markedly and HCC cells were blocked at G2/M phase. Cyclin D1 and CDK1 expression were both decreased but not significantly. Cdc25C expression was increased significantly. PS473 Akt and pT308 Akt expression level was increased significantly as well, which were almost the same with those transfected with LY294002.ConclusionLZTS1 could inhibit HCC cell proliferation by impairing PI3K/Akt pathway.     

原发性肝癌组织cyclinB1表达水平及其临床诊断意义

目的探讨细胞周期蛋白B1(cyclinB1)在肝癌组织的表达及其临床诊断意义。方法用逆转录聚合酶链反应(RTPCR)检测40例原发性肝细胞癌患者癌组织及其癌旁组织cyclinB1mRNA的表达。结果90%(36/40)癌组织及85%(34/40)癌旁组织cyclinB1mRNA呈阳性表达,癌组织cyclinB1mRNA表达量为0.531±0.015,明显高于癌旁组织0.263±0.023,两者比较有统计学意义(P<0.05)。cyclinB1mRNA表达与肝癌的病理分级、淋巴转移与否有关,而与性别、年龄、肿瘤大小、癌栓形成与否无关。结论在肝癌发生、发展中,cyclinB1可能起着重要作用,cyclinB1表达水平可作为肝癌分级、分化及癌转移的辅助衡量指标之一。     

uPA和P-P38在肝癌组织中的表达及相关性研究

目的 探讨尿激酶型纤溶酶原激活剂(uPA)和磷酸化P38(P-P38)在原发性肝癌(PHC)中的表达及其意义.方法 采用免疫组织化学S-P法检测34例原发肝癌组织、14例癌旁正常肝组织中uPA和P-P38的表达情况.结果 原发性肝癌组织中uPA和P-P38表达定位于细胞浆和细胞核,其表达明显高于癌旁正常肝组织(P＜0.05).二者的表达与性别、年龄、肿瘤大小等没有相关性(P＞0.05),与淋巴结转移、组织分化及癌栓等有关(P＜0.05),二者之间比较表达呈明显正相关(P＜0.05).结论 二者在原发性肝癌呈高表达,且与原发性肝癌的发生、发展和转移密切相关.