CN117398478B - Mkp5在糖尿病肾病治疗中的应用 - Google Patents
Mkp5在糖尿病肾病治疗中的应用 Download PDFInfo
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- CN117398478B CN117398478B CN202311713853.5A CN202311713853A CN117398478B CN 117398478 B CN117398478 B CN 117398478B CN 202311713853 A CN202311713853 A CN 202311713853A CN 117398478 B CN117398478 B CN 117398478B
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- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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Abstract
本发明属于生物医学技术领域,公开了MKP5在糖尿病肾病治疗中的应用,SC‑MKP5‑PLGA‑PEI纳米粒制剂在治疗糖尿病肾病的应用。本发明提供糖尿病肾病治疗新靶点和仿生纳米载体,优先或主动地靶向哺乳动物肾组织并递送MKP5,基因表达的转录后调控的相应蛋白表达。组织特异性靶向通过使用具有适合靶向锚的纳米载体来实现,该靶向锚为干细胞膜。为糖尿病肾病基因治疗打下坚实基础。本发明的靶向递送策略将选择性地递送MKP5质粒至肾组织,保护正常组织免受给药的治疗剂的损害。此类策略应通过增加治疗剂的治疗指标,同时降低最小治疗剂量进而从降低治疗风险方面来提高治疗效果。
Description
技术领域
本发明属于生物医学技术领域,尤其涉及MKP5在糖尿病肾病治疗中的应用。
背景技术
糖尿病肾病(Diabetic nephropathy, DN)是糖尿病重要的并发症之一,也是导致终末期肾病的主要原因。糖尿病肾病致病因素多样,包括早期临床症状不明显,呈慢性进行性发展且病理改变复杂等,难以进行及时有效的临床治疗,更缺乏有效的治疗手段。糖尿病肾病典型病理表现为肾小球体积增大、细胞外基质过度沉积、系膜区域扩张、管状基底膜增厚等,随着病情加重导致肾纤维化,包括肾小管间质纤维化及肾小球硬化,最终发展为肾衰竭,临床治疗效果有限,病死率高。糖尿病肾病的发生和发展涉及多途径和介质参与,包括多种细胞因子、氧化应激和炎症反应等作用过程。随着糖尿病发病率升高,糖尿病肾病群体逐年扩大,进一步明确糖尿病肾病的病理生理机制,探究有效治疗靶点十分必要。
丝裂原活化蛋白激酶磷酸酶5 [Mitogen-activated protein kinase (MAPK)phosphatases 5, MKP5] 是MAPK家族的主要负调控磷酸酶。MKP5基因可参与细胞自噬、凋亡、炎症和氧化应激等多种生理病理过程,参与调控TGF-β/Smad、MAPK、AKT等信号通路,在肺纤维化、心脏纤维化和肥胖相关的糖尿病等疾病中均发挥重要作用。但是关于MKP5在糖尿病肾病和肾纤维化中的治疗作用尚未见报道。因此,探究MKP5基因在DN中的作用对于DN的基因治疗至关重要。然而,游离核酸药物体内全身给药使得裸露的核酸分子不稳定且容易被核酸酶降解,结构过于亲水亦不利于其进入细胞发挥疗效,它们在体内还会引起毒副作用,例如免疫系统的激活和凝血时间的延长等。需借助一定的递送系统。
目前常用的核酸递送载体为病毒、脂质体和仿生纳米粒。病毒载体已广泛用于miRNA递送,并且在多种细胞类型上有较高的转染效率。然而,其安全问题阻碍了基于病毒载体治疗的临床应用,如:包装上限(粒径20-40 nm)递送核酸分子量限制严格(通常<5kb)、插入风险、肝毒性、免疫原性等。核酸疫苗快速发展以来,脂质载体引起了人们的广泛关注。但目前其缺乏靶向性、脱靶率高、免疫反应、脂质纳米颗粒诱导的细胞毒性等问题,依然没有很好的解决。因此,需要开发更安全、有效和靶向的纳米递送平台。
通过上述分析,现有技术存在的问题及缺陷为:DN的分子机制尚未完全了解,急需寻找有效作用靶点;MKP5在DN中的作用不清楚,需确定MKP5与DN的关系;由于糖尿病肾病目前临床治疗障碍,因此迫切需要有靶向递送策略;还需要开发更安全、有效和靶向的纳米递送平台。
发明内容
针对现有技术存在的问题,本发明首先公开了一种MKP5在糖尿病肾病治疗中的应用。
本发明公开了一种MKP5在糖尿病肾病治疗中的应用;所述MKP5干细胞膜仿生纳米粒为干细胞膜包载的MKP5-PLGA-PEI纳米粒,所述纳米粒为球形结构。
所述MKP5为改善糖尿病肾病的有效作用靶点。
所述干细胞膜为实现细胞特异性靶向的靶向锚。
所述载MKP5干细胞膜仿生纳米粒优先或主动地靶向哺乳动物细胞种类并递送用于基因表达的转录后调控的工具(DNA)。
封装DNA用于在肾细胞中进行翻译或表达的肾细胞特异性纳米载体。
靶向递送策略选择性地递送MKP5质粒至肾组织。
靶向递送策略增加治疗剂的治疗指标,同时降低最小治疗剂量。
本发明还公开了一种SC-MKP5-PLGA-PEI纳米粒制剂,所述SC-MKP5-PLGA-PEI纳米粒制剂为注射剂。
所述SC-MKP5-PLGA-PEI纳米粒制剂在治疗糖尿病肾病的应用。
第一,本发明提供一种有效的治疗靶点,并运用靶向递送策略将活性试剂特异性递送至特定细胞种类以治疗哺乳动物中的糖尿病肾病的方法。
本发明涉及糖尿病肾病治疗新靶点和仿生纳米载体,也称为纳米药物,以及优先或主动地靶向哺乳动物肾组织并递送MKP5基因,基因表达的转录后调控的相应蛋白表达。组织特异性靶向通过使用具有适合靶向锚的纳米载体来实现,该靶向锚为干细胞膜。为糖尿病肾病基因治疗打下坚实基础。
本发明的靶向递送策略将选择性地递送MKP5质粒至肾组织,保护正常组织免受给药的治疗剂的损害。此类策略应通过增加治疗剂的治疗指标,同时降低最小治疗剂量进而从降低治疗风险方面来提高治疗效果。
本发明确定过表达MKP5可以缓解糖尿病肾病的发生,并提供的SC-MKP5-PLGA-PEI纳米粒制剂在治疗糖尿病肾病的应用,可提高肾保留率,减少非特异性组织分布,可以改善糖尿病肾病小鼠肾小球内的胶原沉积和病理变化,缓解糖尿病肾病小鼠肾脏纤维化。
第二,本发明的技术方案填补了国内外业内技术空白:关于MKP5在糖尿病肾病和肾纤维化中的治疗作用尚未见报道。因此,本研究明确了MKP5在糖尿病肾病中的作用;MKP5在糖尿病肾病中治疗效果目前没有研究,本研究填补了这项空白。
第三,载MKP5干细胞膜仿生纳米粒改善糖尿病肾病的应用具有以下显著的技术进步:
1. 靶向递送策略:该技术利用干细胞膜作为靶向锚,使纳米粒能够优先或主动地靶向肾细胞。这种靶向递送策略增加了治疗剂在糖尿病肾病病灶的富集程度,提高了治疗效果。
2. 干细胞膜包裹:干细胞膜作为纳米粒的包裹材料,具有良好的生物相容性和生物活性,能够增强纳米粒在体内的稳定性和生物活性。同时,干细胞膜还可以提供病变组织细胞的特异性靶向,确保纳米粒主要作用于病变的肾脏组织细胞,减少对其他组织的影响。
3. MKP5的作用靶点:MKP5是改善糖尿病肾病的有效作用靶点。通过将MKP5质粒DNA加载到纳米粒中,并通过靶向递送策略将其优先递送至肾组织,可以实现MKP5的高效表达,从而发挥其对糖尿病肾病的治疗作用。
4. 基因表达调控:该技术可通过转录后调控的机制,调控相关基因的表达。通过封装DNA并递送到肾细胞中,纳米粒可以在肾细胞内实现翻译或表达,从而改变糖尿病肾病的病理过程,实现治疗效果。
总体而言,载MKP5干细胞膜仿生纳米粒改善糖尿病肾病的应用通过靶向递送和基因表达调控等策略,实现了对糖尿病肾病的精确治疗,具有较高的治疗效果和生物安全性,为糖尿病肾病治疗领域带来了显著的技术进步。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对本发明实施例中所需要使用的附图做简单的介绍,显而易见地,下面所描述的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的图1(A) Western blot检测STZ诱导的小鼠肾组织中MKP5蛋白表达水平图;图1(B) 小鼠肾组织MKP5与UACR变化情况图;图1(C) Image J 分析免疫组化中MKP5阳性面积与肾组织系膜面积分数变化情况图( * * * P<0.001 );
图2是本发明实施例提供的形态学水平分析MKP5敲除后糖尿病肾病小鼠肾组织病理改变图;A. Masson染色观察小鼠肾组织中肾小球胶原的沉积图,Image Pro plus软件对胶原表达进行相对定量分析图;B. Sirius red染色观察小鼠肾组织中肾小球胶原的沉积图,Image Pro plus软件对胶原表达进行相对定量分析图;C. HE染色分析小鼠肾组织中肾小球形态学变化图,Image J软件对肾小球面积进行相对定量分析图;D. PAS染色分析小鼠肾组织中肾小球形态学变化图,Image J软件对肾小球面积进行相对定量分析图;每组n=4。*,P<0.05;
图3是本发明实施例提供的MKP5 敲除后小鼠肾组织中纤维化指标的表达变化图;A. Real-time PCR法检测小鼠肾组织中Fibronectin、Collagen IV基因转录水平图(每组n=4);B. Western blot法分析小鼠肾组织中Fibronectin和Collagen IV蛋白表达变化图;C. 使用Image J软件进行条带相对灰度分析图(每组n=6);*,P<0.05;**,P<0.01;
图4是本发明实施例提供的图4(A) SC包被MKP5-PLGA-PEI纳米粒TEM图;图4(B)SC-MKP5-PLGA-PEI纳米粒细胞摄入图;
图5是本发明实施例提供的SC-MKP5-PLGA-PEI纳米粒组织分布图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
以下是本发明提供的两个具体的实施例和它们的实现方案:
实施例1:制备MKP5-PLGA-PEI纳米粒:首先,合成PLGA(聚乳酸-羟基乙酸)和PEI(聚乙烯亚胺)的共聚物作为纳米粒的载体材料。然后,将MKP5质粒DNA加载到纳米粒中,形成MKP5-PLGA-PEI纳米粒。
包裹干细胞膜:将干细胞膜与MKP5-PLGA-PEI纳米粒进行包裹,使纳米粒表面覆盖有干细胞膜。
靶向递送:通过靶向锚的作用,使MKP5-PLGA-PEI纳米粒优先或主动地靶向肾细胞,并递送MKP5质粒DNA。
基因表达调控:MKP5质粒DNA在肾细胞内被释放,通过转录后调控的机制,调控相关基因的表达,从而改善糖尿病肾病的病理过程。
实施例2:制备MKP5-PLGA-PEI纳米粒:采用相同的方法合成MKP5-PLGA-PEI纳米粒。
封装DNA:将用于翻译或表达的DNA(例如,编码特定蛋白质)加载到MKP5-PLGA-PEI纳米粒中。
肾细胞特异性递送:由于纳米粒表面覆盖有干细胞膜,具有细胞特异性锚的作用,使MKP5-PLGA-PEI纳米粒优先靶向肾细胞。
翻译或表达:MKP5-PLGA-PEI纳米粒在肾细胞中释放DNA,并实现翻译或表达,从而发挥MKP5的疗效,改善糖尿病肾病的病理过程。
这些实施例和实现方案仅供参考,具体的制备和应用方法需要根据具体需求进行调整和优化。在实际应用中,还需要进行进一步的研究和临床验证来评估其疗效和安全性。
针对现有技术存在的问题,本发明提供了载(丝裂原活化蛋白激酶磷酸酶5)MKP5干细胞膜仿生纳米粒改善糖尿病肾病的应用。
所述载MKP5干细胞膜仿生纳米粒为干细胞膜包载的MKP5-聚乳酸-羟基乙酸共聚物-聚醚酰亚胺(SC-MKP5-PLGA-PEI)纳米粒,所述纳米粒为球形结构。
所述MKP5为改善糖尿病肾病的有效作用靶点。
所述干细胞膜为实现细胞特异性靶向的靶向锚。
所述载MKP5干细胞膜仿生纳米粒优先或主动地靶向哺乳动物细胞种类并递送用于基因表达的转录后调控的工具(DNA)。
封装DNA用于在肾细胞中分别进行翻译或表达的肾细胞特异性纳米载体。
靶向递送策略为选择性地递送MKP5质粒至肾组织。
靶向递送策略增加治疗剂的治疗指标,同时降低最小治疗剂量。
一种SC-MKP5-PLGA-PEI纳米粒制剂,所述SC-MKP5-PLGA-PEI纳米粒制剂为注射剂。
SC-MKP5-PLGA-PEI纳米粒制剂在治疗糖尿病肾病的应用。
本发明的一些实施例是指将基因药物优先递送至哺乳动物受试者的细胞的
方法,使得仿生纳米载体能够实现由细胞膜受体介导的摄取,以便向所述细胞递送其基因药物负载。
“方法”涉及哺乳动物受试者注射根据本发明的靶向纳米载体,其包含基因药物,用于糖尿病肾病的治疗。
所述工具选自质粒,质粒在整合入宿主细胞基因组时抑或在染色体外时表达基因,因此引发肽的或蛋白的基因产物的翻译。
纳米载体是基于脂质的纳米载体,优选脂质体、脂质复合物、或胶束。纳米载体不止基于脂质,优选该非脂质基纳米载体是合成高分子纳米粒子、树枝状聚合物、碳纳米管、或胶体金纳米粒子。特别优选的是,合成高分子纳米粒子是聚(D,L-乳酸-乙醇酸共聚物)纳米粒子。
靶向锚包括干细胞膜部分,该靶向部分可以是靶向锚经由靶向部分特异性结合在细胞种类的表面上表达的趋向性。
如上所述,当以游离形式给药时,核酸并不是非常有效。因此,它们成功用于治疗疾病需要采用能够使得治疗性核酸安全有效地到达其靶标的纳米载体。因此,本发明人建立了另一种靶向药物递送的模型,即通过将其封装于纳米载体内递送至目的组织。
本发明实施例在研发或者使用过程中取得了一些积极效果,和现有技术相比的确具备很大的优势,下面内容结合试验过程的数据、图表等进行描述。
1. 构建糖尿病肾病小鼠模型
处理组小鼠注射STZ(50 mg/kg/day),对照组小鼠注射等量的柠檬酸缓冲溶液,连续五天腹腔注射。末次注射2 w后,连续2 d检测小鼠空腹血糖,大于198 mg/dl视为糖尿病造模成功。糖尿病小鼠随机分成四组(n=8):WT-Control组、WT-STZ组、KO-Control组、KO-STZ组,继续饲养至20 w。
2. Western blot实验
(1)制胶:按照免封闭PAGE凝胶快速制备试剂盒说明书,取分离胶缓冲液和分离胶溶液各2.7/2.7 mL充分混匀,加入35μL的过硫酸铵溶液混匀,注入制胶玻璃板中。取浓缩胶缓冲液和浓缩胶各0.75/0.75 mL,加入10μL的过硫酸铵溶液,混匀后注入制胶玻璃板中,插入梳齿,凝固后拔去梳齿,用于电泳实验。
(2)电泳:取出适量蛋白样品和蛋白分子量标准品(Marker)加入上样孔。补满新鲜配置的电泳液后,进行电泳。电泳条件:先恒压100 V,约20 min,使样品齐平;样品跑至分离胶区域,改为恒压140 V。
(3)转膜:取适宜大小的PVDF膜,在甲醇溶液中浸泡30 s,使膜激活。在转膜夹上,从正极到负极按照以下顺序放置:海绵、滤纸、PVDF膜、胶、滤纸、海绵。赶净气泡,转膜夹夹紧,放入倒满预冷转膜液的转膜槽中,将转膜槽放入冰水浴中。转膜条件:恒流250 mA,时间为1.5 h。
(4)封闭:取出PVDF膜后,用丽春红染液染色,以蛋白Marker为参照,根据目的蛋白所在位置剪膜。TBST洗10 min,至丽春红颜色残留洗净。将膜放入快速封闭液中,室温震荡封闭1 h。
(5)敷一抗:吸净封闭液,按说明书稀释比例稀释一抗后,加入含PVDF膜的孵育盒中,置于4℃摇床,孵育过夜。
(6)敷二抗:TBST漂洗3次,每次10 min。加入稀释到一定比例的二抗,室温下摇床震荡孵育1 h。
(7)显影:按1:1比例混合A液、B液配置显影液,将待检膜放入显影液中孵育3 min。将PVDF膜放入显影仪中曝光,调节曝光时间,保证条带质量。
3.Real-time PCR实验
3.1 RNA提取
为防止RNA降解和污染,以下实验操作中均使用无RNase的移液器头和EP管。
3.2 提取组织RNA提取
(1)取50mg动物组织样品置于EP管中。加入500 μL Trizol试剂后,使用组织匀浆器匀浆3次。4℃、14000 rpm离心15 min,小心吸取上清至新的EP管中。
(2)加入1/5体积Trizol的氯仿,涡旋震荡,充分混匀后,室温下静置5min。4℃、12000 rpm离心15 min。EP管中溶液分层,总的RNA溶于上层水溶液中。小心吸取上层液体至新的EP管中。
(3)加入与(2)中吸取液体等体积的异丙醇,以沉淀RNA。缓慢颠倒6-8次,使其混匀。室温静置10 min,12000 rpm离心15 min。观察EP管底部出现白色沉淀,倒掉上清。
(4)DEPC处理水配置的75%乙醇预冷后,加入500 μL至EP管,12000 rpm离心15min。吸弃上清,开盖挥发乙醇3 min。加入适量预冷的DEPC处理水,轻轻吸打,以溶解RNA沉淀。
3.3细胞RNA提取
(1)收板后,吸净六孔板内培养液,加入Trizol试剂(500 μL/孔),常温裂解5 min后,反复吹打至细胞完全脱落,转移至1.5 mL EP管中。
(2)加入Trizol的1/5体积的氯仿,后续步骤与提取组织RNA步骤一致。
3.4总RNA浓度测定
使用酶标仪检测260 nm处吸光度,测得样品的RNA浓度,以DEPC处理水为Blank组。计算各样品浓度,并用DEPC处理水调整,使各样品RNA浓度一致。 逆转录反应
(1)按下表配置去除基因组DNA体系,42℃反应2 min。
表4 逆转录反应配置体系
(2)在上述步骤反应液中加入10 μL 2×Hifair® Ⅱ SuperMix plus试剂,按下表参数进行逆转录:表5 逆转录反应参数
(3) Real-time PCR检测
实验步骤参照FastStart Universal SYBR Green Master (ROS) 试剂盒说明书。
1)反应体系:
表6 Real-time PCR反应体系
2)反应参数:
表7 Real-time PCR反应参数
3)采用相对定量法2-△△Ct分析数据,计算目的基因相对于内参基因GAPDH的表达倍数。实验步骤参照FastStart Universal SYBR Green Master (ROS) 试剂盒说明书。
4. 组织染色
4.1石蜡切片制备
新鲜小鼠肾组织在4%多聚甲醛固定液中固定2 d后,依次在梯度酒精中脱水、二甲苯试剂中透明、加热后的石蜡和二甲苯等比例混合的溶液中放置处理30 min、浸入融化的石蜡等其凝固、石蜡包埋机器上包埋形成蜡块、石蜡切片机器上切出组织切片(厚度5 μm)、展片贴于黏附载玻片上烤干,即得石蜡切片。
4.2 HE染色
(1)二甲苯中脱蜡2次,每次15 min;无水乙醇中浸泡2次,每次5 min;90%乙醇、80%乙醇、70%乙醇依次各浸泡5 min,蒸馏水洗3 min。
(2)苏木素染液中染色15 min,蒸馏水洗;伊红染液染色20 s,蒸馏水洗。
(3)95%乙醇快速脱水,无水乙醇脱水3次,每次10 s。放至二甲苯溶液中透明2次,每次2 min。蘸取适量中性树胶,滴加到组织切片上,盖上盖玻片,赶净气泡,室温避光放置。
(4)HE染色切片用光学显微镜拍照后,照片采用Image J软件进行分析,以肾小球面积进行统计(每组n=4,每只取5个视野)。
4.3 PAS 染色
(1)脱蜡、水化操作与HE染色中相同。
(2)氧化剂滴加处理10 min,流水冲洗5 min,甩片去水,擦干切片上残留水分。滴加Schiff染液,染色25 min。流水冲洗5 min后,苏木素复染4 min,酸性乙醇分化,水洗,返蓝,水洗。
(3)脱水、透明、中性树胶封固。
(4)PAS染色切片用光学显微镜拍照后,照片采用Image J软件进行分析,统计肾小球面积(每组n=4,每只取5个视野)。
4.4 Masson 染色
(1)组织石蜡切片脱蜡、水化。Weigert铁苏木素染液按1:1比例现用现配,滴加染色10 min。酸性乙醇分化5 s,蒸馏水洗终止消化。蓝化液返蓝5 min,蒸馏水洗1 min。滴加丽春红染色液染3 min。
(2)蒸馏水与弱酸溶液按1:1比例配制弱酸工作液,洗1 min。磷钼酸溶液洗2 min,再用弱酸工作液洗1 min。苯胺蓝染液染色4 min,弱酸工作液洗1 min。
之后进行脱水、透明、封片。
(3)Masson染色切片用光学显微镜拍照后,照片采用Image J Pro Plus软件进行分析,以胶原沉积面积与肾小球总面积之比进行统计(每组n=4,每只取5个视野)。
4.5 Sirius red染色
(1)脱蜡、水化后,Weigert铁苏木素染液染色20 min,流水冲洗5 min,蒸馏水洗1次。滴加Sirius red染色液,染色80 min。
(2)配置酸化水工作液(酸化水浓缩液:蒸馏水=1:100)洗2次,每次10 s。蒸馏水洗2次。之后进行脱水、透明、封片。
(3)Sirius red染色切片用光学显微镜拍照后,照片采用Image J Pro Plus软件进行分析,以胶原沉积面积与肾小球总面积之比进行统计(每组n=4,每只取5个视野)。
4.6免疫组化
(1)石蜡切片脱蜡并水化,擦干多余水分,免疫组化笔画圈包含组织切片。加入3%H2O2 溶液,室温孵育10 min。消除内源性过氧化物酶活性。PBS工作液洗3次,每次2 min。
(2)配置抗原修复工作液(抗原修复浓缩液用PBS稀释50倍),预热至99℃,并加热保持温度。放切片修复15 min,自然冷却,PBS洗3次。
(3)滴加由PBS配置的5% BSA封闭液,室温封闭30 min。甩去封闭液,敷一抗,放入湿盒中4℃过夜。
(4)过夜后,PBS洗3次,敷二抗,37℃孵育1 h。PBS洗3次,DAB显色液(现用现配)滴加显色2-5 min。镜下观察颜色变化。蒸馏水终止显色。
(5)苏木素复染、酸性乙醇分化,流水冲洗30 min后,PBS洗3次。之后脱水、透明、封片。
(6)免疫组化切片用光学显微镜拍照后,照片采用Image J Pro Plus软件进行分析,以DAB阳性染色面积与总面积之比进行统计(每组n=4,每只取5个视野)。
5. 纳米粒形态学考察
使用TEM(日本东京Jeol,Jeol JEM1230)观察NPs的形态。简言之,在TEM分析之前,将SC-PLGA-PEI和PLGA-PEI分别滴加在碳涂层400目铜网上,然后等其自然挥干。用0.1%磷钨酸溶液染色(PAS)20 min,用滤纸除去多余的PAS后,于透射电镜上拍照。
6.细胞摄入考察
将密度为2×104个/mL 的HMC细胞接种于六孔板中,在5% (体积分数) CO2及37 ℃条件下培养过夜,在对应孔中加入1.0 mL含40μg MKP5的SC-PLGA-PEI孵育12 h后,PBS)冲洗3次,每次1.0 mL,再加入1.0 mL PBS 和2 μL1 mg/mL Hoechest33342 染料染核,静置15min,用倒置荧光显微镜观察拍照。
7. 组织分布
尾静脉注射cy5.5标记的MSC-MKP5-PLGA-PEI纳米药物,并使用IVIS®活体光学系统(Perkinlemer,IVIS Kinetic,MA,USA)(n=4)检测生物分布(640 nm/670 nm)。
结果
为了检测MKP5与糖尿病肾病的关系,构建了链脲佐菌素(Streptozocin, STZ)诱导糖尿病肾病动物模型。通过Western blot法检测STZ诱导后糖尿病肾病小鼠肾组织中MKP5表达变化。结果显示,STZ处理后WT组小鼠肾组织中MKP5 的基因转录水平较WT对照组小鼠显著下调(P<0.05)(图1A)。尿白蛋白/尿肌酐(UACR)、免疫组化和PAS染色结果显示,与对照组相比,STZ诱导组肾组织中MKP5基因转录水平明显降低。与对照组相比,STZ诱导后小鼠的尿白蛋白水平显著上升、UACR值显著增加, STZ诱导后,系膜区域进一步扩增,基底膜也明显增厚(图1B和C)。
糖尿病肾病患者的肾脏在病理学上通常有肾脏肥大、基膜增厚、细胞外基质沉积、肾小球硬化和间质纤维化等表现。细胞外基质蛋白(ECM)包括胶原、层粘连蛋白、纤维连接蛋白和各种蛋白多糖,在高糖、TGF-β等多种因素刺激下可使其生成过量并积聚,ECM沉积则是糖尿病肾病形成的主要因素。为了观察敲除MKP5对糖尿病小鼠肾组织中肾小球的病理学结构改变的作用,本发明通过HE和PAS染色观察肾病小鼠肾脏中肾小球形态学变化,通过Masson染色和Sirius red染色观察小鼠肾脏中肾小球胶原沉积情况。
为了观察MKP5敲除后对糖尿病肾病小鼠肾纤维化程度的影响,进一步通过Masson染色和Sirius red染色检测小鼠肾脏中肾小球胶原沉积情况,分别表现为蓝色和红色结合区域。如图2A和B所示,在正常小鼠肾小球中胶原表达很少,未经STZ诱导的KO组小鼠肾小球中胶原结合面积在Masson染色中也非常少,在Sirius red染色中较正常小鼠有所增加,但无统计学意义。在WT组中,与对照组相比STZ诱导后小鼠肾小球中代表胶原沉积的蓝色和红色结合区域显著增多(P<0.05),表示糖尿病肾病小鼠肾小球中已出现明显的胶原沉积。在STZ诱导组中,KO组小鼠肾小球内蓝色和红色结合区域面积较WT组小鼠进一步显著增加(P<0.05)。表明敲除MKP5会进一步促进STZ诱导的糖尿病肾病小鼠肾小球内的胶原沉积,加剧糖尿病肾病小鼠肾脏纤维化。
HE染色和PAS染色结果如图2C和D示所示,在WT组中,与对照相比,STZ诱导后小鼠肾脏出现了明显的形态学改变,主要表现为肾小球肥大、系膜基质增生、系膜扩张及肾小球基底膜增厚。在STZ诱导组中,KO组小鼠肾小球面积与WT组小鼠相比显著增加,系膜区域进一步扩增,基底膜也明显增厚。对HE和PAS染色结果中的肾小球面积进行统计分析,STZ诱导的WT组小鼠肾小球面积与其对照组相比扩大一倍(P<0.05)。STZ处理后,KO组小鼠肾小球面积较WT小鼠增加约40%(P<0.05)。说明敲除MKP5可加剧糖尿病肾病小鼠肾小球的病理学改变,促进糖尿病肾病的发展。
在糖尿病肾病中,Fibronectin和Collagen IV是ECM的主要成分,通常作为肾纤维化标志性蛋白。Real-time PCR结果如图3A所示,WT小鼠中,STZ诱导后小鼠肾组织中Fibronectin、Collagen IV基因的转录水平与对照组相比显著上升(P<0.01); STZ诱导后,KO组小鼠肾组织中的Fibronectin、Collagen IV基因的转录水平较WT组小鼠进一步显著上调。Western blot结果显示,WT处理组小鼠肾组织中的Fibronectin和Collagen IV蛋白的表达量较WT对照组小鼠显著增加(P<0.01)。STZ处理后,KO组小鼠肾组织中纤维化标志性蛋白的表达与WT组小鼠相比进一步显著增加(P<0.05)(图 3B和C)说明敲除MKP5会加重糖尿病肾病小鼠肾脏纤维化程度。
图4A是MKP5-PLGA-PEI纳米粒的透射电子显微镜直观图,结果显示纳米粒为球形结构, 干细胞膜显示出明显膜结构,干细胞膜包载的MKP5-PLGA-PEI纳米粒膜结构和纳米粒结构清晰可见,同时证明干细胞膜包载的MKP5-PLGA-PEI纳米粒制备成功。
药物能否被载体材料携带进入细胞,是衡量该材料是否是一个良好的药物载体的重要指标之一。通常,药物以内吞方式被细胞摄入,因此药效与细胞内吞摄入量密切相关。为了验证SC-MKP5-PLGA-PEI纳米粒可高效通透细胞膜的假设,采用荧光倒置显微镜初步检测了标记Cy5.5的SC-MKP5-PLGA-PEI纳米粒被HMC 细胞株摄入的情况,结果见图4B , 红色荧光表示药物摄取进入细胞的分布及强度,荧光强度越大摄取量越多。从荧光强度上比较,孵育12h 时, SC-MKP5-PLGA-PEI纳米粒摄入明显强于MKP5-PLGA-PEI纳米粒,是干细胞膜具有较好的生物相容性,增强了纳米粒与细胞的融合,促进细胞的摄取。
还使用糖尿病肾病小鼠模型测定了SC-MKP5-PLGA-PEI纳米粒制剂的组织分布。静脉注射Cy5.5标记的SC-MKP5-PLGA-PEI纳米粒制剂12 h后,使用IVIS®活体成像系统对病变部位和主要要器官进行体外成像(图5)。结果表明,与MKP5-PLGA-PEI纳米粒制剂相比,SC-MKP5-PLGA-PEI纳米粒制剂的肾蓄积显著增加,但肝脏蓄积显著减少。结果表明,SC-MKP5-PLGA-PEI纳米粒制剂显著提高肾保留率,减少非特异性组织分布。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
Claims (2)
1.一种MKP5在制备糖尿病肾病治疗药物中的应用,其特征在于,MKP5质粒DNA载入干细胞膜仿生纳米粒,制成干细胞膜包载的MKP5-PLGA-PEI纳米粒,所述纳米粒为球形结构;所述MKP5质粒DNA为改善糖尿病肾病的有效作用靶点。
2.一种干细胞膜包载的MKP5-PLGA-PEI纳米粒制剂,其特征在于,所述干细胞膜包载的MKP5-PLGA-PEI纳米粒制剂为注射剂。
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