Analysis of Vaginal Microecological in Women with Cervical Intraepithelial Neoplasia and Cervical Cancer
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摘要:
目的 研究宫颈上皮内瘤变和宫颈癌患者阴道微生态特点,为宫颈疾病预防和治疗提供参考。 方法 选取昆明医科大学第三附属医院(云南省肿瘤医院)2018年7月至2018年10月病理诊断为宫颈上皮内瘤变患者(n = 65)、宫颈癌患者(n = 51)和同期体检健康女性(n = 88)为研究对象,收集阴道拭子,通过显微镜镜检进行微生态评价。 结果 与健康组相比,宫颈上皮内瘤变组在菌群密集度构成上,差异有统计学意义(P < 0.05),在菌群多样性、优势菌、pH、Nugent评分构成上,差异无统计学意义(P > 0.05)。宫颈癌组与健康组在菌群密集度、优势菌、pH、Nugent评分构成上,差异均有统计学意义(P < 0.05),在菌群多样性构成上,差异无统计学意义(P > 0.05)。 结论 宫颈上皮内瘤变和宫颈癌患者阴道微生态失调,以宫颈癌失调最为显著。尽早发现阴道微生态失调,恢复阴道微生态平衡,有利于防治宫颈癌。 Abstract:Objective To investigate the characteristics of vaginal microecology in patients with cervical intraepithelial neoplasia and cervical cancer, and provide information for the prevention and treatment of cervical diseases. Methods From July 2018 to October 2018, patients with cervical intraepithelial neoplasia (n = 65), patients with cervical cancer (n = 51) and healthy control (n = 88) in Yunnan Cancer Hospital were selected. Vaginal swabs were collected and microecological assessment were performed by microscopic examination. Results Compared with healthy group, there was a significant difference in the composition of bacterial density in the cervical intraepithelial neoplasia group (P < 0.05), despite there was no significant difference in flora diversity, dominant bacteria, pH and Nugent score (P > 0.05). There were significant differences in flora density, dominant bacteria, pH and Nugent score composition between the cervical cancer group and the healthy group (P < 0.05) and there was no significance in the composition of bacterial diversity (P > 0.05). Conclusion Patients with cervical intraepithelial neoplasia and cervical cancer have microecological imbalance, especially in cervical cancer patients. Early detection and restoration of female vaginal microecological imbalance are beneficial for the prevention and treatment of cervical cancer. -
接种疫苗被认为是世界范围内预防感染最经济的措施。一种有效的佐剂对于提高疫苗接种效率是非常必要的。脂质体是磷脂双分子层包裹水相而构成的类球状微囊,按电荷性质可分为中性脂质体、阴离子脂质体和阳离子脂质体[1]。其中阳离子脂质体比阴离子和中性脂质体更有效,可延长在注射部位抗原的停留时间,增加抗原提呈,并诱导更强的免疫反应[2-4]。
DC-Chol(3β-[N-(N′ ,N′ -二甲基氨基乙烷)-氨基甲酰基])是胆固醇衍生物,含有一个叔胺基团。DC-Chol毒性相对较小[5],通常与脂质二油酰磷脂酰乙醇胺(DOPE)结合使用[6-7]。胆固醇是经典脂质体配方的主要成分,被阳离子衍生物(DC-Chol)取代,形成PLUSCOM[8],可有效吸附抗原[9-10]。ISCOMs作为佐剂,以多种方式增强免疫反应,通过抗原提呈细胞对微粒优先摄取,PLUSCOM在诱导抗原特异性CD8 T细胞反应方面与经典ISCOMs一样有效[11]。
本研究以四价流感病毒裂解疫苗原液作为模型药物,探讨DC-Chol修饰脂质体作为载体对该疫苗的免疫增强效果。研究中选择市售疫苗原液和PBS作为对照组,比较DC-Chol脂质体作为疫苗佐剂的免疫增强效果。同时还对DC-Chol脂质体中DC-Chol用量与免疫原性的量效关系进行了初步研究,为阳离子脂质体佐剂的开发奠定基础。
1. 材料与方法
1.1 材料
1.1.1 流感疫苗
均由江苏沃森生物技术有限公司提供。H1N1批号SA2018002,H3N2批号SB2018002,B(V)批号SC2018001,B(Y)批号SC22018006。
1.1.2 实验动物
SPF级昆明种小鼠,雌性,6~8周龄,体重18~22 g,由昆明医科大学实验动物中心提供[合格证号为SCXK9(滇)2005-0008]。
1.1.3 主要试剂
大豆卵磷脂(北京美亚斯磷脂技术公司);胆固醇(北京鼎国昌盛生物技术有限责任公司);DC-Cholesterol(Avanti Polar Lipids,USA);MTT(北京博奥拓达科技有限公司);Anti-mouse CD4 PE、Anti-mouse CD8a FITC(eBioscience,USA)。
1.2 方法
1.2.1 DC-Chol脂质体的制备
采用薄膜分散法和冻融-冻干法[12]。将胆固醇(80 mg)和大豆磷脂(300 mg)溶于无水乙醇,减压旋转成膜;在水化的脂质体混悬液中加入DC-Chol水浴静置,加入一定量流感疫苗原液,制备脂质体冻干粉。
1.2.2 DC-Chol脂质体包封率检测
高速离心取上清液,通过Lowry蛋白法[12]计算包封率。
1.2.3 DC-Chol脂质体量效关系研究
小鼠随机分为七组,每组3只,不同剂量DC-Chol脂质体组(250、500、750、900 μg/只)、PBS组、疫苗原液组、中性脂质体组,抗原剂量为6 μg/只。腹腔免疫后第7天处死,通过MTT法[13]测定刺激指数(SI)确定最佳DC-Chol剂量。
1.2.4 DC-Chol脂质体细胞免疫原性研究
在方法1.2.3确定最佳用量的基础上制备脂质体进行免疫实验。小鼠随机分为PBS组、疫苗原液组、中性脂质体组、DC-Chol 脂质体组,每组9只,腹腔免疫,于第7天、14天、28天处死,MTT方法检测各组SI值,流式细胞术检测T淋巴细胞表面标记。
1.3 统计学处理
采用SPSS17.0软件进行统计分析,多组间比较通过单因素方差分析,以P < 0.05 为差异有统计学意义。
2. 结果
2.1 DC-Chol 脂质体包封率结果
蛋白含量测定的标准曲线为Y = 0.0032X - 0.0009,相关系数R2 = 0.9984,在10~100 μg/mL 范围内有良好线性关系。DC-Chol流感疫苗脂质体包封率结果,见表1。
表 1 不同含量的DC-Chol阳离子脂质体的包封率Table 1. Encapsulation efficiency of DC-Chol cationic liposomes with different contentsDC-Chol含量(µg/鼠) 包封率(%) 250 59.17 500 70.44 750 68.78 900 68.78 2.2 DC-Chol 脂质体量效关系研究
检测结果显示,与PBS组、疫苗原液组相比较,剂量分别为250、500、750、900 μg的DC-Chol组差异有统计学意义(P < 0.05),表明DC-Chol脂质体有较好的免疫原性,见图1;500、750、900 μg组三个剂量组间比较差异无统计学意义(P > 0.05),选择500 µg/鼠为DC-Chol修饰脂质体疫苗的最佳用量。
2.3 DC-Chol 修饰流感疫苗脂质体细胞免疫原性研究
2.3.1 脾淋巴细胞增殖实验
DC-Chol脂质体组与中性脂质体组、疫苗原液组、PBS组比较差异有统计学意义(P < 0.05),且SI值高于各组,说明DC-Chol阳离子脂质体能有效刺激脾淋巴细胞增殖,产生较早较强的免疫原性,增强细胞免疫,见图2。DC-Chol脂质体组7 d时刺激小鼠脾淋巴细胞增殖的强度最大,诱导细胞免疫的水平最高,但其14~28 d SI值稍有上升,说明抗原刺激机体时产生的抗体不会一直存在于机体中,部分会通过以代谢或排泄的方式排出体外,但仍然有细胞免疫原性的存在即记忆细胞。
2.3.2 T淋巴细胞表面标记实验
由图3可知,DC-Chol脂质体组与中性脂质体组、疫苗原液组、PBS组比较差异有统计学意义(P < 0.05),说明DC-Chol阳离子脂质体可增强细胞免疫;免疫相同周期时,DC-Chol组28 d与14 d的CD4+/CD8+值进行比较差异有统计学意义(P < 0.05),随着时间的延长,DC-Chol修饰的脂质体疫苗对脾淋巴细胞的刺激强度增加,有延长免疫时间的作用。
3. 讨论
阳离子脂质体已成为新一代的疫苗佐剂和给药系统。Yifan Ma[14]通过制备不同表面电荷密度的阳离子脂质体,作用于C57小鼠,采用 ELISA方法和流式细胞术发现阳离子脂质体能诱导更强的免疫反应,证实了阳离子脂质体的免疫调节作用主要是由于其表面电荷密度,而不是阳离子脂质体的浓度。Brunel等[15]将DC-Chol用于乙型肝炎疫苗,结果表明DC-Chol具有免疫调节作用,能诱导BALB/c小鼠的Th1和Th2型免疫反应。Rui等[16]开发了一种由肺炎球菌表面蛋白a和阳离子DC-Chol脂质体组成的肺炎球菌鼻腔疫苗,用小鼠肺炎链球菌感染模型验证了该疫苗的有效性。DC-Chol脂质体能同时诱导体液免疫和细胞免疫,诱导产生IgGl和IgG2a;DC-Chol脂质体还能诱导粘膜免疫[17-18]。阳离子脂质体能够运载不同种类的药物或作为疫苗载体,且到目前人们仍然不断开发其应用潜力。阳离子脂质体的毒性在一定程度上限制了它的应用,未来需要更加深入研究其结构和作用机制,设计出更加低毒高效的阳离子。
本实验中制备的DC-Chol脂质体疫苗包封率均在50% 以上。选择PBS、市售流感疫苗原液以及中性脂质体作为对照组,在一个免疫周期内DC-Chol脂质体的SI值始终高于其他组,提示 DC-Chol流感疫苗脂质体冻干粉在体内可产生细胞免疫,延长免疫时间,具有明显的佐剂效果。该实验为今后研究DC-Chol脂质体佐剂提供了初步参考,未来还需对其作用机制和安全性方面深入研究。
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表 1 健康组、宫颈上皮内瘤变组阴道微生态比较[n(%)]
Table 1. Comparison of vaginal microecology between healthy control and cervical intraepithelial neoplasia group [n(%)]
项目 健康组(n = 88) 宫颈上皮内瘤变组(n = 65) χ2/Z P 菌群密集度 未见/Ⅰ级 40(45.45) 44(67.69) −2.641 0.009* Ⅱ/Ⅲ级 48(54.55) 20(30.77) Ⅳ级 0(0.00) 1(1.54) 菌群多样性 −0.867 0.386 未见/Ⅰ级 74(84.09) 58(89.23) Ⅱ/Ⅲ级 14(15.91) 6(9.23) Ⅳ级 0(0.00) 1(1.54) 优势菌 革兰阳性大杆菌 62(70.45) 52(80.00) 5.174 0.159 革兰阳性球菌 4(4.55) 2(3.08) 革兰阴性短杆菌 1(1.14) 3(4.62) 无 21(23.86) 8(12.30) pH 3.8~4.5 51(57.95) 47(73.31) 3.345 0.067 > 4.6 37(42.05) 18(27.69) Nugent评分(分) 0~3 54(56.81) 46(70.77) −1.366 0.172 4~6 30(34.09) 19(29.23) > 7 4(9.09) 0(0.00) 与健康组比较,*P < 0.05。 表 2 健康组、宫颈癌组阴道微生态比较[n(%)]
Table 2. Comparison of vaginal microecology between healthy control and cervical cancer group [n(%)]
项目 健康组(n = 88) 宫颈癌组(n = 51) χ2/Z P 菌群密集度 未见/Ⅰ级 40(45.45) 36(70.59) −2.859 0.004* Ⅱ/Ⅲ级 48(54.55) 15(29.41) Ⅳ级 0(0.00) 0(0.00) 菌群多样性 未见/Ⅰ级 74(84.09) 45(88.24) −0.816 0.537 Ⅱ/Ⅲ级 14(15.91) 5(9.80) Ⅳ级 0(0.00) 1(1.96) 优势菌 革兰阳性大杆菌 62(70.45) 23(45.10) 10.543 0.014* 革兰阳性球菌 4(4.55) 8(15.69) 革兰阴性短杆菌 1(1.14) 2(3.92) 无 21(23.86) 18(35.29) pH 3.8~4.5 51(57.95) 10(19.61) 19.28 < 0.001* > 4.6 37(42.05) 41(80.39) Nugent评分(分) 0~3 54(61.36) 17(33.33) −2.948 0.003* 4~6 30(34.09) 32(62.75) > 7 4(0.05) 2(3.92) 与健康组比较,*P < 0.05。 -
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