中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2010年
8期
603-608
,共6页
韩凌斐%邱伟民%胡程%王玲%姚红霞%熊诗诣%孟梦%方勇%马丁
韓凌斐%邱偉民%鬍程%王玲%姚紅霞%熊詩詣%孟夢%方勇%馬丁
한릉비%구위민%호정%왕령%요홍하%웅시예%맹몽%방용%마정
宫颈肿瘤%受体,免疫%HSP70热休克蛋白质类%疱疹病毒科%疾病模型,动物
宮頸腫瘤%受體,免疫%HSP70熱休剋蛋白質類%皰疹病毒科%疾病模型,動物
궁경종류%수체,면역%HSP70열휴극단백질류%포진병독과%질병모형,동물
Uterine cervical neoplasms%Receptors,immunologic%HSP70 heat-shock proteins%Herpesviridae%Disease models,animal
目的 探讨B、T淋巴细胞弱化因子(BTLA)胞外段联合热休克蛋白(HSP)70-宫颈癌TC-1细胞(HSP70-TC-1)抗原肽复合物治疗小鼠宫颈癌模型的效果.方法 (1)将TC-1细胞接种于C57BL/6小鼠成瘤后,实时荧光定量PCR技术检测小鼠肿瘤组织中BTLA、疱疹病毒侵入介质(HVEM)mRNA的表达,流式细胞仪检测肿瘤组织中浸润的淋巴细胞细胞膜表面BTLA、HVEM的表达(以荧光强度表示).(2)小鼠接种TC-1细胞后,根据处理方法的不同分为5组,即pcDNA3.1(注射空载体pcDNA3.1质粒,作为对照)、psBTLA(注射表达BTLA胞外段的真核表达载体psBTLA质粒)、HSP70(注射HSP70-TC-1抗原肽复合物)、HSP70+pcDNA3.1(注射HSP70-TC-1抗原肽复合物和pcDNA3.1质粒)、HSP70+psBTLA(注射HSP70-TC-1抗原肽复合物和psBTLA质粒)组,观察各组小鼠体内肿瘤的生长情况;并采用实时荧光定量PCR技术检测各组肿瘤组织中相关免疫基因包括γ干扰素(IFN-γ)、白细胞介素(IL)2、IL-10、转化生长因子β(TGF-β)和人叉头型基因P3(FoxP3)mRNA的表达;免疫组化法计数各组瘤旁组织中CD8+T淋巴细胞;7-氨基放线菌素D/羧基荧光素乙酰乙酸琥珀酰亚胺酯双标法检测各组脾淋巴细胞的杀伤效应(以杀伤率表示);氚标记胸腺嘧啶核苷掺入法测定各组脾淋巴细胞的增殖活性,酶联免疫吸附试验测定各组脾淋巴细胞培养上清液中IL-2和IFN-γ的浓度.结果 (1)小鼠接种TC-1细胞后第7、14、21、28天,其肿瘤组织中BTLA mRNA的表达水平逐渐上调,第14天达最高,为2.83±0.35,与第7天的1.66±0.25比较,差异有统计学意义(P<0.05);而HVEM mRNA的表达水平无明显变化(P>0.05).接种TC-1细胞后第7、14天,肿瘤组织中浸润的淋巴细胞细胞膜表面BTLA的平均荧光强度分别为33.5和51. 8,两者比较,差异有统计学意义(P<0.05);而HVEM的平均荧光强度分别为57.2和49.3,两者比较,差异无统计学意义(P>0.05).(2)接种后第14、28天,HSP70+psBTLA组抑瘤率分别为65%、88%,均明显高于其他组(P<0.05).接种后第28天,HSP70+psBTLA组IFN-γ、IL-2 mRNA的相对表达水平(分别为3.12±0.71、3.20±0.62)高于其他组,TGF-β、IL-10和Foxp3 mRNA的相对表达水平(分别为0.25±0.03、0.31±0.04、0.19±0.03)低于其他组,分别比较,差异均有统计学意义(P<0.05).HSP70+psBTLA组瘤旁组织中CD8+T淋巴细胞数为(52±6)个/高倍视野,脾淋巴细胞对TC-1细胞的杀伤率为(65.5±2.4)%,脾淋巴细胞的增殖活性为15.0×103 cpm,IL-2和IFN-γ的浓度分别为(824±51)、(1096±112)pg/ml,均明显高于其他组(P<0.05).结论 真核表达载体psBTLA表达的BTLA胞外段联合HSP70-TC-1抗原肽复合物可使肿瘤微环境中的免疫相关基因表达更有利于抗肿瘤免疫应答,对TC-1细胞构建的小鼠宫颈癌模型具有很好的治疗效果.
目的 探討B、T淋巴細胞弱化因子(BTLA)胞外段聯閤熱休剋蛋白(HSP)70-宮頸癌TC-1細胞(HSP70-TC-1)抗原肽複閤物治療小鼠宮頸癌模型的效果.方法 (1)將TC-1細胞接種于C57BL/6小鼠成瘤後,實時熒光定量PCR技術檢測小鼠腫瘤組織中BTLA、皰疹病毒侵入介質(HVEM)mRNA的錶達,流式細胞儀檢測腫瘤組織中浸潤的淋巴細胞細胞膜錶麵BTLA、HVEM的錶達(以熒光彊度錶示).(2)小鼠接種TC-1細胞後,根據處理方法的不同分為5組,即pcDNA3.1(註射空載體pcDNA3.1質粒,作為對照)、psBTLA(註射錶達BTLA胞外段的真覈錶達載體psBTLA質粒)、HSP70(註射HSP70-TC-1抗原肽複閤物)、HSP70+pcDNA3.1(註射HSP70-TC-1抗原肽複閤物和pcDNA3.1質粒)、HSP70+psBTLA(註射HSP70-TC-1抗原肽複閤物和psBTLA質粒)組,觀察各組小鼠體內腫瘤的生長情況;併採用實時熒光定量PCR技術檢測各組腫瘤組織中相關免疫基因包括γ榦擾素(IFN-γ)、白細胞介素(IL)2、IL-10、轉化生長因子β(TGF-β)和人扠頭型基因P3(FoxP3)mRNA的錶達;免疫組化法計數各組瘤徬組織中CD8+T淋巴細胞;7-氨基放線菌素D/羧基熒光素乙酰乙痠琥珀酰亞胺酯雙標法檢測各組脾淋巴細胞的殺傷效應(以殺傷率錶示);氚標記胸腺嘧啶覈苷摻入法測定各組脾淋巴細胞的增殖活性,酶聯免疫吸附試驗測定各組脾淋巴細胞培養上清液中IL-2和IFN-γ的濃度.結果 (1)小鼠接種TC-1細胞後第7、14、21、28天,其腫瘤組織中BTLA mRNA的錶達水平逐漸上調,第14天達最高,為2.83±0.35,與第7天的1.66±0.25比較,差異有統計學意義(P<0.05);而HVEM mRNA的錶達水平無明顯變化(P>0.05).接種TC-1細胞後第7、14天,腫瘤組織中浸潤的淋巴細胞細胞膜錶麵BTLA的平均熒光彊度分彆為33.5和51. 8,兩者比較,差異有統計學意義(P<0.05);而HVEM的平均熒光彊度分彆為57.2和49.3,兩者比較,差異無統計學意義(P>0.05).(2)接種後第14、28天,HSP70+psBTLA組抑瘤率分彆為65%、88%,均明顯高于其他組(P<0.05).接種後第28天,HSP70+psBTLA組IFN-γ、IL-2 mRNA的相對錶達水平(分彆為3.12±0.71、3.20±0.62)高于其他組,TGF-β、IL-10和Foxp3 mRNA的相對錶達水平(分彆為0.25±0.03、0.31±0.04、0.19±0.03)低于其他組,分彆比較,差異均有統計學意義(P<0.05).HSP70+psBTLA組瘤徬組織中CD8+T淋巴細胞數為(52±6)箇/高倍視野,脾淋巴細胞對TC-1細胞的殺傷率為(65.5±2.4)%,脾淋巴細胞的增殖活性為15.0×103 cpm,IL-2和IFN-γ的濃度分彆為(824±51)、(1096±112)pg/ml,均明顯高于其他組(P<0.05).結論 真覈錶達載體psBTLA錶達的BTLA胞外段聯閤HSP70-TC-1抗原肽複閤物可使腫瘤微環境中的免疫相關基因錶達更有利于抗腫瘤免疫應答,對TC-1細胞構建的小鼠宮頸癌模型具有很好的治療效果.
목적 탐토B、T림파세포약화인자(BTLA)포외단연합열휴극단백(HSP)70-궁경암TC-1세포(HSP70-TC-1)항원태복합물치료소서궁경암모형적효과.방법 (1)장TC-1세포접충우C57BL/6소서성류후,실시형광정량PCR기술검측소서종류조직중BTLA、포진병독침입개질(HVEM)mRNA적표체,류식세포의검측종류조직중침윤적림파세포세포막표면BTLA、HVEM적표체(이형광강도표시).(2)소서접충TC-1세포후,근거처리방법적불동분위5조,즉pcDNA3.1(주사공재체pcDNA3.1질립,작위대조)、psBTLA(주사표체BTLA포외단적진핵표체재체psBTLA질립)、HSP70(주사HSP70-TC-1항원태복합물)、HSP70+pcDNA3.1(주사HSP70-TC-1항원태복합물화pcDNA3.1질립)、HSP70+psBTLA(주사HSP70-TC-1항원태복합물화psBTLA질립)조,관찰각조소서체내종류적생장정황;병채용실시형광정량PCR기술검측각조종류조직중상관면역기인포괄γ간우소(IFN-γ)、백세포개소(IL)2、IL-10、전화생장인자β(TGF-β)화인차두형기인P3(FoxP3)mRNA적표체;면역조화법계수각조류방조직중CD8+T림파세포;7-안기방선균소D/최기형광소을선을산호박선아알지쌍표법검측각조비림파세포적살상효응(이살상솔표시);천표기흉선밀정핵감참입법측정각조비림파세포적증식활성,매련면역흡부시험측정각조비림파세포배양상청액중IL-2화IFN-γ적농도.결과 (1)소서접충TC-1세포후제7、14、21、28천,기종류조직중BTLA mRNA적표체수평축점상조,제14천체최고,위2.83±0.35,여제7천적1.66±0.25비교,차이유통계학의의(P<0.05);이HVEM mRNA적표체수평무명현변화(P>0.05).접충TC-1세포후제7、14천,종류조직중침윤적림파세포세포막표면BTLA적평균형광강도분별위33.5화51. 8,량자비교,차이유통계학의의(P<0.05);이HVEM적평균형광강도분별위57.2화49.3,량자비교,차이무통계학의의(P>0.05).(2)접충후제14、28천,HSP70+psBTLA조억류솔분별위65%、88%,균명현고우기타조(P<0.05).접충후제28천,HSP70+psBTLA조IFN-γ、IL-2 mRNA적상대표체수평(분별위3.12±0.71、3.20±0.62)고우기타조,TGF-β、IL-10화Foxp3 mRNA적상대표체수평(분별위0.25±0.03、0.31±0.04、0.19±0.03)저우기타조,분별비교,차이균유통계학의의(P<0.05).HSP70+psBTLA조류방조직중CD8+T림파세포수위(52±6)개/고배시야,비림파세포대TC-1세포적살상솔위(65.5±2.4)%,비림파세포적증식활성위15.0×103 cpm,IL-2화IFN-γ적농도분별위(824±51)、(1096±112)pg/ml,균명현고우기타조(P<0.05).결론 진핵표체재체psBTLA표체적BTLA포외단연합HSP70-TC-1항원태복합물가사종류미배경중적면역상관기인표체경유리우항종류면역응답,대TC-1세포구건적소서궁경암모형구유흔호적치료효과.
Objective To investigate the synergistic therapy effects of B and T lymphocyte attenuator(BTLA) extracellular domain in combination with heat shock protein 70 (HSP70)-TC-1 antigen peptide complex on the mouse model of cervical cancer and the related immunological mechanisms. Methods(1)Detecting the BTLA and herpesvirus entry mediator (HVEM) gene expression in the tumor microenvironment after C57BL/6 mice were inoculated with TC-1 tumor cells by realtime PCR; BTLA,HVEM expression on tumor infiltrating lymphocytes cell surface were detected by flow cytometry (fluorescence intensity). (2) According to different treatments, tumor-bearing mice were divided into 5 groups, which was injected with pcDNA3. 1 (empty vector plasmid as control), psBTLA (vector plasmid which expresses BTLA extracellular domain), HSP70 (HSP70-TC-1 cell peptide complex), HSP70 +pcDNA3.1 or HSP70 + psBTLA, respectively. The weight of tumor was recorded. The expression of immunoregulatory genes in tumor microenvironment were detected. The change of lymphocyte amount and cytotoxicity were detected too; lymphocyte proliferation activity was measured by tritium thymidine incorporation assay; the concentration of interleukin (IL) 2 and interferon-γ(IFN-γ) in supernatants of spleen lymphocyte were measured by enzyme-linked immunosorbent assay (ELISA). Results (1) BTLA gene expression was gradually increased after tumor cells inoculation. The highest expression level was 2. 83 + 0. 35 at 14th day, which had statistical significance difference with the 7th day expression of 1.66±0. 25 (P < 0. 05). While HVEM mRNA expression did not change significantly (P > 0. 05). The 7th and 14th day after TC-1 cells inoculation, the average fluorescence intensity of BTLA expression on the surface of tumor infiltrating lymphocytes was 33.5 and 51.8, respectively, in which there was statistically significant difference (P <0. 05); while the difference of HVEM expression was not statistically significant (57. 2 vs 49. 3 ,P >0. 05). (2)The 28th day after inoculation, tumor inhibition rate of HSP70 + psBTLA group was 88%, which was significantly higher than other treatment groups (P <0. 05). The 28th day after TC-1 cells inoculation, combination therapy not only promoted IFN-γ and IL-2 gene (3. 12 + 0.71,3.20 + 0. 62)expression but also reduced transforming growth factor-β (TGF-β), Foxp3 and IL-10 expression (0. 25±0. 03,0. 19 +0. 03,0. 31 +0. 04;P <0. 05). It also promoted CD8+ T lymphocyte infiltration(52 +6)/high power field, cytotoxicity (65.5±2.4) %, proliferation (15.0 × 103 cpm) and cytokine IL-2 , IFN-γsecretion(824±51), (1096±112) pg/ml, which were all significantly higher than other groups (P <0. 05). Conclusion The effect of immunotherapy on tumor can be augmented by the combination of psBTLA which expresses extracellular domain of BTLA and HSP70-TC-1 tumor antigen peptide complex,which could improve the expression of the related immunoregulatory genes to establish a much better microenvironment in favor of anti-tumor immune response against the mice model of the cervix carcinoma.
