CN114836420A - Periostin特异性干扰质粒和PD-1特异性干扰质粒的组合及其应用 - Google Patents
Periostin特异性干扰质粒和PD-1特异性干扰质粒的组合及其应用 Download PDFInfo
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Abstract
本发明公开了Periostin特异性干扰质粒和PD‑1特异性干扰质粒的组合及其应用。本发明利用RNAi技术从基因表达源头上进行阻断Periostin和PD‑1的产生，利用减毒沙门氏菌运载Periostin特异性干扰质粒和PD‑1特异性干扰质粒来治疗结直肠癌，将为临床上结直肠癌的治疗提供新的策略及制剂。
Description
技术领域
本发明涉及Periostin特异性干扰质粒和PD-1特异性干扰质粒的组合及其应用。属于生物技术领域。
背景技术
结直肠癌在全球范围内都是影响程度较大的恶性肿瘤，发病率在恶性肿瘤中排名第三，死亡率排名第四。我国结直肠癌发病率位居恶性肿瘤第四位，死亡率位居第五位。但是近年来，结直肠癌发病率和死亡率在全世界范围内均呈上升趋势。目前，临床治疗结直肠癌仍以手术、放化疗、靶向药为主，但总体效果差强人意。
细胞外基质蛋白Periostin与多种疾病的发生、发展密切相关。大量的研究表明细胞外基质蛋白Periostin能够影响组织再生、炎症、纤维化以及肿瘤的发生发展。Periostin蛋白可通过活化PI3K/Akt信号通路增强肿瘤血管生成和肿瘤细胞存活进而促进结直肠癌细胞肝转移，并且Periostin依赖的YAP活化则可导致肿瘤细胞分泌IL-6因子进而促进成纤维细胞的活化及Periostin蛋白表达，这种由组织微环境因子Periostin和IL-6介导的肿瘤细胞与基质细胞相互作用共同促进肠炎相关肠癌的发生。
免疫逃逸是肿瘤的一大特征，细胞程序性死亡受体-1(PD-1)和细胞程序性死亡受体-配体1(PD-L1)是导致肿瘤免疫逃逸的重要免疫检查点分子，阻断PD-1/PD-L1可重新激活细胞毒性T细胞对肿瘤细胞的杀伤作用。因此，针对免疫检查点分子PD-1或PD-L1的免疫疗法成为近年研究的热点。有研究表明，PD-1/PD-L1抑制剂能够显著改善部分结直肠癌患者预后，但是还有部分结直肠癌患者不能受益，且会有不良反应及耐受现象。肿瘤微环境中的免疫抑制细胞、细胞因子、免疫抑制靶点等组成了一道“墙”，阻碍T细胞接近肿瘤细胞发挥杀伤作用。在一些肿瘤微环境中，T细胞未成功接近肿瘤细胞，肿瘤细胞可能存在“保护层”，T细胞不能抵达肿瘤细胞发挥杀伤肿瘤作用。在肿瘤微环境中，肿瘤微环境中，Periostin能够促进成纤维细胞的活化和血管生成，它在癌细胞的“保护层”形成过程中可能扮演了重要的角色，推测其可能造成了癌细胞与T细胞的隔离。通过靶向Periostin可以将先前的“冷”肿瘤(无反应、无免疫活性的肿瘤)转变为“热”肿瘤(易受宿主免疫系统攻击的肿瘤)。Periostin抑制剂可能会增强PD-1等免疫检查点抑制剂治疗的成功率，这样的组合用药疗法会为更多的患者带来希望。
发明内容
本发明的目的是为克服上述现有技术的不足，提供一种Periostin特异性干扰质粒和PD-1特异性干扰质粒的组合及其应用。
为实现上述目的，本发明采用下述技术方案：
1、Periostin特异性干扰质粒在制备抗结直肠癌药物中的应用。
2、一种抗结直肠癌试剂盒，包含减毒沙门氏菌运载Periostin特异性干扰质粒。
3、PD-1特异性干扰质粒的组合在制备抗结直肠癌药物中的应用。
4、一种抗结直肠癌试剂盒，包含减毒沙门氏菌运载PD-1特异性干扰质粒。
5、Periostin特异性干扰质粒和PD-1特异性干扰质粒的组合。
6、Periostin特异性干扰质粒和PD-1特异性干扰质粒的组合在制备抗结直肠癌药物中的应用。
7、一种抗结直肠癌试剂盒，包含Periostin特异性干扰质粒和PD-1特异性干扰质粒，它们分别利用减毒沙门氏菌运载。
优选的，所述的Periostin特异性干扰质粒，是将Periostin特异性干扰序列(其核苷酸序列为：5’-GGAAATTGTAAGAGCCACC-3’)通过常规酶切连接入pll3.7质粒(上海吉凯基因化学有限公司)而得。
优选的，所述的PD-1特异性干扰质粒，是将PD-1特异性干扰序列(其核苷酸序列为：5’-G A T C C G G G T T T G A G C C A A C C C G T C C A G T T C A A G A G AC T G G A C G G GTTGGCTCAAACCTTTTTTGGAAA-3’)(新乡医学院基础医学院提供)通过常规酶切连接入pGCSilencer neo2.0-U6质粒(上海吉凯基因化学有限公司)而得。
优选的，所述减毒沙门氏菌为phoP/phoQ减毒沙门氏菌。
本发明的有益效果：
本发明利用RNAi技术从基因表达源头上进行阻断Periostin和PD-1的产生，利用减毒沙门氏菌运载Periostin特异性干扰质粒和PD-1特异性干扰质粒来治疗结直肠癌，将为临床上结直肠癌的治疗提供新的策略及制剂，并产生重要经济效益。
在诸多肿瘤基因治疗的载体中，申请人选择了毒性相对小，且转染效率相对较高的第三类载体——细菌性载体。经基因改造的减毒沙门氏菌菌株phoP/phoQ菌株，能够选择性地在肿瘤中定居和繁殖，并且能够将质粒DNA转染到真核细胞中，同时对正常组织和器官的副作用非常小。
沙门氏菌作为Periostin和PD-1特异性干扰质粒运载具有如下优点：
(1)沙门氏菌具有在肿瘤组织中聚集的特性：与正常组织相比较，在瘤组织内可高出5000到10000倍。
(2)沙门氏菌的细胞内侵袭特性使其可在巨噬细胞内存活：这与其在血液和体液内的转移和运输稳定性和对实体肿瘤的靶向性密切相关。
(3)沙门氏菌为兼性厌氧菌使其即可在高度缺氧的较大实体肿瘤内生长：又可在有氧的较小的转移瘤内存活。
附图说明
图1为本发明实施例的小鼠结直肠癌使用本发明的试剂盒治疗和其他对照组治疗结束之后收取的肿瘤样本，检测本发明试剂盒抑制肿瘤微环境中Periostin和PD-1的表达情况。
图2为本发明实施例的小鼠结直肠癌使用本发明的试剂盒治疗和其他对照组治疗结束之后收取的肿瘤样本拍照结果对比。
图3为本发明实施例的小鼠结直肠癌使用本发明的试剂盒治疗和其他对照组治疗结束之后各组肿瘤重量统计分析结果。
图4为本发明实施例的小鼠结直肠癌使用本发明的试剂盒治疗和其他对照组治疗结束之后各组肿瘤微环境中免疫细胞浸润比例的改变。
图5为本发明实施例的小鼠结直肠癌使用本发明的试剂盒治疗和其他对照组治疗结束之后各组肿瘤微环境中免疫细胞浸润比例统计分析结果。
具体实施方式
下面结合附图和实施例对本发明进行进一步的阐述，应该说明的是，下述说明仅是为了解释本发明，并不对其内容进行限定。
实施例：
1、减毒沙门氏菌运载Periostin特异性干扰质粒(pSi-Periostin)的制备：
(1)制备phoP/phoQ减毒沙门氏菌(由新乡医学院基础医学院提供)感受态细胞；感受态细胞的制备方法参照常规分子生物学实验进行，本申请不予赘述；
(2)通过电转化的方式将Periostin特异性干扰质粒转化入phoP/phoQ减毒沙门氏菌感受态细胞(1：200)，涂布Amp+LB固体板子，挑取单克隆，即获得减毒沙门氏菌运载Periostin特异性干扰质粒。
其中，所述的Periostin特异性干扰质粒，是将Periostin特异性干扰序列(其核苷酸序列为：5’-GGAAATTGTAAGAGCCACC-3’)通过常规酶切连接入pll3.7质粒(上海吉凯基因化学有限公司)而得。Periostin特异性干扰序列为申请人自行制备(Yang,Y.etal.Periostin deficiency attenuates lipopolysaccharide and obesity-inducedadipose tissue fibrosis.FEBS Letters 595,2099-2112,doi:10.1002/1873-3468.14154(2021))。
2、减毒沙门氏菌运载PD-1特异性干扰质粒(pSi-PD-1)
(1)制备phoP/phoQ减毒沙门氏菌(由新乡医学院基础医学院提供)感受态细胞；感受态细胞的制备方法参照常规分子生物学实验进行，本申请不予赘述；
(2)通过电转化的方式将PD-1特异性干扰质粒转化入phoP/phoQ减毒沙门氏菌感受态细胞(1：200)，涂布Amp+LB固体板子，挑取单克隆，即获得减毒沙门氏菌运载PD-1特异性干扰质粒。
其中，所述的PD-1特异性干扰质粒，是将PD-1特异性干扰序列(其核苷酸序列为：5’-G A T C C G G G T T T G A G C C A A C C C G T C C A G T T C A A G A G A CT G G A C G G GTTGGCTCAAACCTTTTTTGGAAA-3’)(新乡医学院基础医学院提供)通过常规酶切连接入pGCSilencer neo2.0-U6质粒(上海吉凯基因化学有限公司)而得。
3、小鼠动物实验
结直肠癌小鼠模型构建：使用6-7周龄C57Bl/6雄鼠，皮下注射1×106MC38结直肠癌细胞，约5-7天成瘤。选用5-7天成瘤大小均一的结直肠癌小鼠30只，分为5组，每组6只，分别为PBS组，Scramble组，pSi-Periostin组，pSi-PD-1组，pSi-Periostin+pSi-PD-1组。
将运载pSi-PD-1和pSi-Periostin的减毒沙门氏菌phoP/phoQ菌(新乡医学院基础医学院提供)接种到100ml的LB培养基(Amp，终浓度为100μg/ml)中，37℃培养过夜(12-16h)。次日早上以1：100再次接种到100ml的LB培养基中，待OD600达0.6时，立即放在冰上。此时沙门氏菌的浓度为1-1.2×109CFU/ml。利用倍比稀释法(用0.1mol/L、pH7.2的PBS)将每组的沙门氏菌稀释到2×106CFU/ml，放置于冰上。进行动物实验，动物分为五组：PBS组，Scramble组，pSi-Periostin组，pSi-PD-1组，pSi-Periostin+pSi-PD-1组。小鼠接种肿瘤细胞后7天，Scramble组每只小鼠注射8×105CFU减毒沙门氏菌运载Scramble质粒(新乡医学院基础医学院提供)，pSi-Periostin组每只小鼠注射8×105CFU减毒沙门氏菌运载pSi-Periostin，pSi-PD-1组每只小鼠注射8×105CFU减毒沙门氏菌运载pSi-PD-1，pSi-Periostin+pSi-PD-1组每只小鼠注射4×105CFU减毒沙门氏菌运载pSi-Periostin和4×105CFU减毒沙门氏菌运载pSi-PD-1，PBS组注射100μl PBS(0.1mol/L、pH7.2)。7天后再次加强注射一次。治疗结束一周之后分离小鼠的肿瘤组织。检测小鼠的肿瘤大小，肿瘤组织中PD-1和Periostin的表达及CD8分子的表达。
在构建结束结直肠癌模型之后的第三周收样，分离小鼠的肿瘤组织。分别制成石蜡切片，65℃烤片2小时，然后常规梯度脱蜡复水，并在蒸馏水中暂时停留；配制0.01M的柠檬酸钠缓冲液(pH＝6.0)并用高火煮沸；将已复水的玻片及玻片架放入缓冲液中，立即调到中火1分钟；然后保温9分钟；将完成抗原修复的玻片拿到室温让其缓慢降温，大约需要5h；将玻片从缓冲液中拿出，用PBS洗10分钟3次；用组化笔在组织周围画圈，切记圈要画得稍大一点，并稍甩干组织后立即加A液(质量浓度3％过氧化氢液)，室温15分钟；用PBS洗玻片10分钟×3次；加B液(山羊血清)封闭，室温1小时；小心吸去封闭液，加一抗，一般抗体适合的浓度为1：200(用含质量浓度0.02％TritonX-100的PBS稀释抗体)；4℃过夜，注意玻片要放平于湿盒中杂一抗；用含质量浓度0.1％Tween-20的PBS洗玻片洗3次，每次5分钟，然后用不含有Tween-20的PBS洗玻片3次，每次5分钟；加二抗C液(鼠兔通用)，室温1小时；洗二抗，操作同洗一抗；加D液，室温20min；PBS洗3次，每次10分钟；然后DAB染色。一般蛋白的染色时间为50秒为最宜，DAB染色结束立即置于自来水中终止反应，然后将玻片浸在蒸馏水中待后续染核；苏木素染核9s，蓝化并梯度脱水，依次经过体积浓度70％、80％、95％、100％的酒精，各2分钟，最后过2道二甲苯，每道10分钟；用中性树胶封片，室温保存切片。
在构建结束结直肠癌模型之后的第三周收样，分离小鼠的肿瘤组织。通过不同密度的percoll进行密度梯度离心获取白细胞，每组的离心管内吸取1×106白细胞悬液至新的ep管内，进行CD45、CD3、CD8、PD-1四染(抗体：PBS＝1：200稀释)，并且设置单染对照，阴性；在4℃冰箱里避光环境孵育30-60分钟。开始设置3500rpm转速，离心4分钟，弃上清。用预冷的1mL PBS进行洗涤细胞，离心步骤同前。每个样品以400μL PBS重悬后，开始检测并分析。
4、实验结果
申请人发现使用减毒沙门氏菌运载pSi-Periostin联合减毒沙门氏菌运载pSi-PD-1的试剂盒治疗结直肠癌小鼠，与其他对照组相比可以更有效的抑制结直肠癌肿瘤的生长。
在构建结束结直肠癌模型之后的第三周收样，分离小鼠的肿瘤组织。图1结果从左到右依次是PBS组、Scramble组、pSi-Periostin组、pSi-PD-1组、pSi-Periostin+pSi-PD-1组，免疫组化结果显示使用减毒沙门氏菌运载pSi-Periostin可以有效抑制肿瘤微环境中Periostin蛋白的表达，并且使用减毒沙门氏菌运载pSi-PD-1可以有效抑制肿瘤微环境中PD-1蛋白的表达，减毒沙门氏菌运载pSi-Periostin联合减毒沙门氏菌运载pSi-PD-1组Periostin和PD-1蛋白表达最低。
图2是各组结直肠癌小鼠肿瘤组织的照片，从上到下依次是PBS组、Scramble组、pSi-Periostin组、pSi-PD-1组、pSi-Periostin+pSi-PD-1组，使用减毒沙门氏菌运载pSi-Periostin联合减毒沙门氏菌运载pSi-PD-1组相比于其他组肿瘤大小显著减小，图3是统计分析图2的结果。
在构建结束结直肠癌模型之后的第三周分离小鼠的肿瘤组织，通过密度梯度离心获取肿瘤浸润的免疫细胞。图4是小鼠动物实验各组肿瘤浸润的免疫细胞中CD8+T细胞的比例，其中，图4从左到右依次是PBS组、Scramble组、pSi-Periostin组、pSi-PD-1组、pSi-Periostin+pSi-PD-1组，pSi-Periostin+pSi-PD-1组的肿瘤浸润CD8+T细胞的比例最高，图5是统计分析图4的结果。
通过上述一系列实验证明，减毒沙门氏菌运载pSi-Periostin联合减毒沙门氏菌运载pSi-PD-1的试剂盒具有很好治疗结直肠癌的效果。目前，临床上还没有应用同时抑制Periostin和PD-1的试剂盒治疗结直肠癌，减毒沙门氏菌运载pSi-Periostin联合减毒沙门氏菌运载pSi-PD-1的试剂盒具有很好的临床应用前景，这是申请人抢先进入的最佳时期。
上述虽然结合附图对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
Claims (7)
1.Periostin特异性干扰质粒在制备抗结直肠癌药物中的应用。
2.一种抗结直肠癌试剂盒，其特征在于，包含减毒沙门氏菌运载Periostin特异性干扰质粒。
3.PD-1特异性干扰质粒的组合在制备抗结直肠癌药物中的应用。
4.一种抗结直肠癌试剂盒，其特征在于，包含减毒沙门氏菌运载PD-1特异性干扰质粒。
5.Periostin特异性干扰质粒和PD-1特异性干扰质粒的组合。
6.Periostin特异性干扰质粒和PD-1特异性干扰质粒的组合在制备抗结直肠癌药物中的应用。
7.一种抗结直肠癌试剂盒，其特征在于，包含Periostin特异性干扰质粒和PD-1特异性干扰质粒，它们分别利用减毒沙门氏菌运载。
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