CN113995852A - Arg-脂质体微囊、微囊包封的鱼精蛋白-siRNA复合体及其制备方法和应用 - Google Patents
Arg-脂质体微囊、微囊包封的鱼精蛋白-siRNA复合体及其制备方法和应用 Download PDFInfo
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- CN113995852A CN113995852A CN202111312542.9A CN202111312542A CN113995852A CN 113995852 A CN113995852 A CN 113995852A CN 202111312542 A CN202111312542 A CN 202111312542A CN 113995852 A CN113995852 A CN 113995852A
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Abstract
本发明提供了一种Arg‑脂质体微囊、微囊包封的鱼精蛋白‑siRNA复合体及其制备方法和应用。Arg‑脂质体微囊是在脂质体微囊悬浮液中加入精氨酸制备而成。微囊包封的鱼精蛋白‑siRNA复合体是将鱼精蛋白‑siRNA复合体包封于Arg‑脂质体微囊中,所述siRNA为针对酪氨酸酶基因的TYR‑siRNA或针对黑色素合成相关MITF基因的MITF‑siRNA。本发明的Arg‑脂质体微囊稳定性高,可用作生物医学药物相转变递送材料。微囊包封的鱼精蛋白‑siRNA复合体可应用于护肤喷剂、面膜或医用敷料,能够局部治疗雀斑、老年斑、黑色素瘤等皮肤问题,对人体其他正常组织安全无害。
Description
技术领域
本发明涉及药物载体技术领域,具体地说是一种Arg-脂质体微囊、微囊包封的鱼精蛋白-siRNA复合体及其制备方法和应用。
背景技术
药物递送系统是指在空间、时间及剂量上全面调控药物在生物体内分布的技术体系,其研究对象包括药物本身和担载药物的载体材料。为担载不同属性的药物,可对药物或载体等进行物理化学改性、修饰。它能够更精准地将药物输送到靶部位,且实现对药物的信号控制释放,克服传统给药方式对正常细胞的毒副作用以及在血液循环中药物释放不可控和药物的生物利用率低等一系列缺点。常见的药物输送材料主要为脂质体(即磷脂),脂质体是指由两亲磷脂双分子层在水中自组装成的一层或多层的球形囊泡结构,大小为几纳米至微米不等。脂质体既可以包封大量的亲水性抗癌药物,还可以包载疏水性药物。与其他纳米级递送载体相比,脂质体作为药物递送载体可以体现出良好的生物相容性和较高的药物包载量,表现在毒性更低,无免疫原性,无致热原性,能够通过正常代谢而被清除。
然而,目前报道的脂质体大多难以解决动力学稳定性问题,致使脂质体容易聚沉,不易长久保存。近年来微针、电渗析等大量的物理促渗技术与脂质体的联合研究用于开发相应物理响应性脂质体。这种方式既能够更好地发挥药效,又能够避免药物注射或口服制剂对人体造成的副作用。但是,由于其价格较高,操作难度较大,限制了其大规模应用。因此研究一种简单的,且能解决脂质体的动力学不稳定性的载体具有重要的现实意义。
基于表观遗传机制的药物通过稳定的靶向载体递送至病变部位是一种新型治疗模式,能够在分子水平上调控异常基因的表达,从而实现基因沉默。氨基酸或蛋白质,是人体必需的物质成分,可用于护肤品、护发剂等。用于提高皮肤免疫功能、调节酸碱、平衡油脂,活化细胞,促进肌肤的新陈代谢和血液循环,对皮肤温和,具有补水保湿、抗皱增白等作用,能够很好的被皮肤吸收。因此,如何将表观遗传机制的药物与护肤成分相结合,研制一种新型的护肤美容产品或治疗产品,无疑是一大创新。
发明内容
本发明的目的就是提供一种Arg-脂质体微囊、微囊包封的鱼精蛋白-siRNA复合体及其制备方法和应用,以解决现有技术中脂质体载体的动力学不稳定性问题,同时,提供一种新型的皮肤美容或治疗的复合体药物。
本发明的目的是这样实现的:一种Arg-脂质体微囊,其以磷脂酰胆碱和磷脂酰丝氨酸为原料,采用薄膜分散法制备脂质体微囊,然后在脂质体微囊悬浮液中加入精氨酸,即得所述Arg-脂质体微囊。
所述磷脂酰胆碱与磷脂酰丝氨酸的质量比为3-5∶1;精氨酸与脂质体微囊的质量比为0.72-0.73:1,上述的Arg-脂质体微囊在药物载体中的应用。
将所述Arg-脂质体微囊作为药物相转变递送载体,Arg-脂质体微囊的相转变温度为41-43℃。
上述的Arg-脂质体微囊的制备方法,包括以下步骤:
a、将磷脂酰胆碱和磷脂酰丝氨酸溶于氯仿中,其中,磷脂酰胆碱与磷脂酰丝氨酸的质量比为3-5∶1,充分混合均匀后,用氮气吹干氯仿,从而形成薄膜状的脂质体;
b、将所得薄膜状的脂质体置于真空干燥器中干燥0.5-2小时,干燥完全后,加入4-羟乙基哌嗪乙磺酸缓冲液,使脂质体分散悬浮,室温静置0.5-2小时;
c、在脂质体悬浮液中加入精氨酸,其中,精氨酸与脂质体微囊的质量比为0.72-0.73:1,混合均匀后,在冰浴条件下,用细胞破碎仪超声分散,即得所述Arg-脂质体微囊。
步骤b中,所述4-羟乙基哌嗪乙磺酸缓冲液的浓度为20mM,pH=7.4;步骤c中,超声功率为80-100W,超声时间为5-10min。
一种微囊包封的鱼精蛋白-siRNA复合体,其将鱼精蛋白-siRNA复合体包封于上述的Arg-脂质体微囊中,所述siRNA为针对酪氨酸酶基因的TYR-siRNA或针对黑色素合成相关MITF基因的MITF-siRNA,所述TYR-siRNA的序列如SEQ:ID:NO:1所示,所述MITF-siRNA的序列如SEQ:ID:NO:2所示。
上述的微囊包封的鱼精蛋白-siRNA复合体的制备方法,包括以下步骤:
a、将siRNA溶液与鱼精蛋白溶液加入到4-羟乙基哌嗪乙磺酸缓冲液中,充分混合均匀后,室温孵育20-40min,从而得到鱼精蛋白-siRNA复合体;其中,鱼精蛋白与siRNA的质量比为12-18∶1;
b、将制备好的鱼精蛋白-siRNA复合体与Arg-脂质体微囊混合,经超声分散后,即得所述微囊包封的鱼精蛋白-siRNA复合体。
步骤a中,所述4-羟乙基哌嗪乙磺酸缓冲液的浓度为20mM,pH=7.4;步骤b中,超声功率为80-100W,超声时间为5-10min。
上述的微囊包封的鱼精蛋白-siRNA复合体在护肤品喷剂、面膜或医用敷料中的应用。
本发明的Arg-脂质体微囊稳定性高,Arg的正电胍基与磷脂的磷酸头部能够发生静电结合和双齿氢键作用形成类似“共价键”的稳定“线性序列结构”,即结构域(如图4所示),实验证实,当Arg浓度为3.0mM时,体系达到最佳稳定状态,其电位值为-50mV左右。本发明的Arg-脂质体微囊可作为一种热敏性药物载体,在42.37℃时显示相变温度,可以用作生物医学药物相转变递送材料。将Arg-磷脂微囊应用于担载鱼精蛋白-siRNA复合体,包载后的Arg-磷脂微囊仍呈球状且分布均匀。此时载药Arg-磷脂微囊的D(90%)和Zeta电位值分别为318.96±4.23nm和-40.59±3.19mV,Arg具有生物相容性,且安全无害,同时解决了脂质体的动力学不稳定性的问题。
本发明的微囊包封的鱼精蛋白-siRNA复合体可应用于护肤喷剂、面膜或医用敷料,依据包封药物不同,应用领域不同。其可局部治疗雀斑、老年斑、黑色素瘤等皮肤问题,对人体其他正常组织安全无害。
本发明的Arg-脂质体包封鱼精蛋白-siRNA复合体,在基因转录水平上使TYR的表达降低,减少黑色素合成。在生物技术领域有广阔的应用前景。Arg-脂质体具有生物相容性,可用于人体皮肤问题的治疗。更重要的是两者皆安全可靠,对正常细胞没有毒副作用,有利于摆脱对放疗、化疗的依赖,从而消除现有技术治疗皮肤问题的缺陷。
附图说明
图1是Arg-脂质体包封鱼精蛋白-siRNA复合体的制备原理示意图。
图2是Arg-脂质体微囊的SEM图。其中,图(a)脂质体微囊;(b)Arg-脂质体微囊。
图3是Arg-脂质体微囊的相变温度图。
图4是精氨酸与脂质体结合的原理示意图。
图5是TYR-siRNA、MITF-siRNA对A375细胞TYR mRNA相对表达量结果图。其中,(a)TYR-siRNA对A375细胞TYR mRNA的相对表达量影响图(*P<0.05);(b)A375细胞TYR蛋白的相对表达量(*P<0.05,n=3);(c)为统计学图(P<0.05);(d)不同浓度TYR-siRNA2对TYRmRNA的影响(*与control相比,P<0.05;#与100pmol相比,P<0.05)(e)不同浓度MITF-siRNA对MITF mRNA的影响(注:*与control相比,P<0.05;#与100pmol相比,P<0.05;**与200pmol相比,P<0.05)(f)不同浓度MITF-siRNA对TYR mRNA的影响(注:*与control相比,P<0.05;#与100pmol相比,P<0.05;**与200pmol相比,P<0.05)。
图6是不同浓度的TYR-siRNA2、MITF-siRNA对细胞凋亡、细胞周期的影响图,其中,(a)(c)分别为不同浓度的TYR-siRNA2、MITF-siRNA对细胞凋亡的影响;(b)(d)分别为不同浓度的TYR-siRNA2、MITF-siRNA对细胞周期的影响。
图7是鱼精蛋白-siRNA复合体TEM图(b)和AFM图(c)(鱼精蛋白与siRNA质量比为15∶1)。
图8是包封鱼精蛋白-siRNA的Arg-脂质体微囊的共聚焦图。
具体实施方式
下面结合具体实施例对本发明的技术方案进行详细说明。本发明实施例中未提到的试验条件和操作均按本领域常规方法或制造商建议的条件进行。所用试剂未表明规格的均为试剂纯或化学纯。
实施例1:Arg-脂质体微囊的制备
1、首先用氯仿溶解磷脂酰胆碱(PC)、磷脂酰丝氨酸(PS),配成浓度分别为25mg/mL、10mg/mL的储备液。采用薄膜分散法将PC、PS按质量比为3-5∶1混合,混匀仪混匀。然后用氮气吹干氯仿,使其在离心管底部成一层薄膜,然后置于玻璃真空干燥器中真空干燥1小时,使氯仿充分挥发。干燥完全后,加入5mL HEPES(20mM,pH=7.40)缓冲液,使脂质体分散悬浮,室温静置1小时。
2、通过加入氨基酸来调整脂质体的稳定性。在脂质体(PC:PS=4:1)悬浮液中加入氨基酸,氨基酸的种类分别为谷氨酸、精氨酸、胍基乙酸、赖氨酸,每种氨基酸设置不同的浓度,分别为0mM、1.5mM、3.0mM、5.25mM、7.5mM、10.0mM(每组脂质体的浓度均为0.72mg/mL),混匀仪混匀后,用细胞粉碎仪超声,将细胞破碎仪的超声探头放置于离心管中央,冰浴。超声功率90W,超声时间8min,使脂质体颗粒大小均匀规整。
3、测定上述体系的粒径及Zeta电位值。将样品置于比色皿和电位槽中,用90PlusPALS粒度仪分别测定不同种类、不同浓度氨基酸-脂质体微囊体系的粒径及Zeta电位值。结果发现,Arg-脂质体微囊的稳定性能最佳,当体系中Arg浓度为3.0mM时,D90%为170.48±0.97nm,电位达到最大值-50.02±1.22mV。
将制备好的Arg-脂质体微囊进行温度响应表征,采用差示扫描量热法(DSC)测定相变温度,结果如图3所示,3.0mM Arg-脂质体的相变温度为42.37℃左右。其中,发现响应温度因加入Arg的含量不同而发生变化,通过扫描电镜(SEM)观察Arg-脂质体的表面形貌(如图2所示)为类球形且形貌规整,可以用作生物医学药物相转变递送材料。
实施例2干预酪氨酸酶基因表达的靶点的确定
2.1、细胞培养
2.1.1细胞培养条件:将黑色素瘤A375细胞培养于含有10%胎牛血清的高糖DMEM培养基(含100U/mL青霉素链霉素双抗,pH 7.2-7.4)中,置于设定为37℃、5%的CO2培养箱中进行常规培养。
2.1.2细胞的换液:当细胞培养液呈现为黄颜色时,弃去原培养液,用PBS缓冲液冲洗细胞1-2遍,加入含有10%胎牛血清的高糖DMEM培养基。
2.1.3细胞的传代:当培养瓶中细胞生长状态较好,铺满瓶底部面积约70%~90%时,进行传代。将原培养液弃去,用PBS缓冲液洗涤细胞2次后,加含0.02%EDTA的0.25%的胰蛋白酶消化细胞。在显微镜下观察,当发现细胞回缩变圆后,弃去胰蛋白酶,加入含有10%胎牛血清的高糖DMEM培养基终止消化,并将贴壁的细胞吹打下来,形成细胞悬液。按照1/3或1/4的比例进行传代。
2.1.4细胞的冻存:按照上述细胞传代步骤,将细胞消化成细胞悬液,放入离心管中。以1000rpm/min转速离心5min,弃去培养基。加入冻存液(以DMEM培养基、胎牛血清、DMSO按照7:2:1比例配制而成)重悬细胞,轻轻吹打细胞,使细胞均匀悬浮,取1.5mL放入冻存管中,封口膜封口做好标记。使用梯度缓慢冻存法冻存细胞:4℃放置10min,-20℃放置30min,-80℃过夜后,将冻存管放入液氮罐中保存。
2.2、siRNA转染
2.2.1设计TYR靶点的siRNA
在NCBI(National Center for Biotechnology Information)基因数据库中搜索Tyrosinase,并从中选择出正确的基因,找到mRNA序列号,最终得到mRNA基因序列。根据mRNA基因序列,设计三条siRNA链,将三条TYR-siRNA和MITF-siRNA作为实验对象,研究不同浓度的TYR-siRNA、MITF-siRNA对A375细胞TYR表达和凋亡、周期的影响,筛选抑制效果最好的干扰片段,实验所用的siRNA均由上海吉玛制药技术有限公司合成。siRNA的序列如表1所示。
表1:
2.2.2siRNA的配制
siRNA开盖前,用12000rpm/min离心1min,防止siRNA开盖散失。慢慢打开管盖,加入125μL的DEPC水,盖上盖后充分震荡混匀,放入-20℃冰箱中冷藏贮存。
2.2.3细胞分组与转染
取对数生长期的黑色素瘤A375细胞,根据上述细胞的传代方法传代接种于六孔板中,将接种的细胞浓度调整为5×105个每孔,于37℃、5%的CO2培养箱中进行常规培养,待细胞贴壁并生长程度约为50%时,进行转染实验。实验分组为:正常对照组、阴性对照组(NC-si RNA)、si RNA1组、si RNA2组、si RNA3组。
根据Lipo High脂质体高效转染试剂说明书,准备转染工作液:
1.将100pmol si RNA1、si RNA2、si RNA3、NC-si RNA分别溶于250μL无双抗的DMEM培养液中稀释,将其轻轻混匀,室温静置5min。
2.取5μL的Lipo High脂质体溶于250μL无双抗的DMEM培养液中稀释,将其轻轻混匀,室温静置5min。
3.将含有Lipo High脂质体的稀释液加入到含有si RNA的稀释液中,轻轻混匀,室温静置10min。
4.将上述转染工作液静置时,用PBS缓冲液洗涤细胞2次,并更换成不含双抗的DMEM培养液。
5.将上述转染工作液逐滴滴入细胞中,轻轻混匀后置于37℃,5%的CO2培养箱中进行培养。
6. 8h后,更换为含有血清的新鲜培养基,置于37℃,5%的CO2培养箱中继续培养。
7. 48h后,收集细胞待后续实验处理。
2.3实时荧光定量PCR筛选特异TYR-si RNA
2.3.1各个基因的引物
在NCBI的基因数据库中,根据人的TYR、β-actin的m RNA序列,运用Primer 5软件设计出一对合适的引物,由生工生物工程股份有限公司合成。
表2:TYR引物序列
2.3.2细胞总RNA的提取
TRIZOL法提取黑色素瘤A375细胞的总RNA,具体步骤为:
1.按照细胞转染的实验操作方法,转染细胞,置于37℃,5%的CO2培养箱中培养48h。
2.将转染的细胞培养48h后,用PBS缓冲液洗涤细胞两遍,每孔加1mL TRIZOL进行裂解,静置3min,用移液枪反复吹打,直至细胞完全裂解后,转移到1.5mL的无RNA酶EP管中,室温放置5-10min,使得核蛋白和核酸完全分离。
3.将上述EP管中加入1/5氯仿(约0.2mL),涡旋振荡器上剧烈振荡混匀15s,室温放置3min。
4.在4℃,12000rpm高速离心机中离心10min,分离出总RNA。小心吸取含有细胞总RNA的上层水相转移至新的1.5mL无RNA酶的EP管中。
5.将上层水相中加入等体积(约0.5mL)异丙醇,上下颠倒混匀数次后,室温条件下静置20min。在4℃,12000rpm高速离心机中离心10min,此时管底可见胶状RNA沉淀。
6.小心吸弃上清,加入75%乙醇(75%乙醇用DEPC水配制)1mL洗涤沉淀。在4℃,12000rpm高速离心机中离心3min,小心弃去上清,保证RNA沉淀无损失。室温干燥5-10min。
7.加入30μL-50μL RNase-free dd H2O,充分溶解RNA。将所得到的RNA溶液置于-80℃冰箱保存或用于后续实验。
2.3.3RNA定量
以DEPC水为对照孔,每孔加入2μL。实验孔分别加入RNA样品2μL,每组设置两个复孔。用酶标仪进行核酸定量,检测各组RNA浓度及其OD值。
2.3.4反转录成c DNA
根据试剂盒说明书,首先去除基因组DNA,按照以下成分于冰上配制反应混合液(1μgRNA样品):
表3:去除基因组DNA混合液的配制
运用PCR仪,设定程序为:42℃2min,4℃2min。反应完成进行下一步。
反转录反应按照以下成分,于冰上配制反应液:
表4:反转录反应液的配制
试剂 | 使用量(μL) |
步骤1的反应液 | 10.0 |
Rnase Free d H2O | 4.0 |
5×Prime Script Buffer2(for Rea L Time) | 4.0 |
RT Primer Mix | 1.0 |
Prime Script RT Enzyme Mix 1 | 1.0 |
运用PCR仪,设定程序为:37℃ 15min,85℃ 5s,4℃ 5min。反应完成后的c DNA分别存放于-80℃。
2.3.5Real Time PCR
以上述步骤得到的c DNA为扩增模板,选择荧光染料SYBR进行实时荧光定量PCR实验,测得每组Ct值。以β-actin为内参,用各组基因的Ct值减去内参的Ct值得到ΔCt值,利用2-△△ct算出每组对应基因的相对表达量。运用SPSS19.0统计软件对基因的表达情况进行统计学分析。注意操作过程均在冰上进行操作。
表5:Real Time PCR反应体系的配制
试剂 | 使用量(μL) |
灭菌水 | 6.0 |
SYBR Premix Ex TaqⅡ | 10.0 |
ROX Reference Dye | 0.4 |
PCR Forward Primer | 1.0 |
PCR Reverse Primer | 1.0 |
RT反应液(c DNA溶液) | 1.0 |
根据试剂盒的反应条件如下:Stage 1,95℃预变性30s;Stage 2,95℃变性5s,55℃退火30s,72℃延伸30s,一共40次循环。选择延伸阶段采集荧光信号。
2.4Western blot检测TYR-si RNA对TYR蛋白表达的影响
2.4.1细胞蛋白提取
1.将实验分组为正常对照组、NC-Si RNA组、TYR-Si RNA组,按照上述的实验操作方法,转染细胞,置于37℃,5%CO2培养箱中培养48h。
2.将六孔板培养液全部弃去,用PBS缓冲液冲洗细胞2遍,每孔加入200μL预冷的含1m M PMSF的RIPA裂解液,置于冰上裂解5min,用细胞刮将六孔板内的细胞刮下,置于EP管中,并放入冰上裂解30min,在此期间每隔10min用涡旋混匀仪震荡1次,每次10s。
3.裂解后,在4℃,14000rpm高速离心机中离心10min,取上清。
2.4.2细胞蛋白定量及变性
用考马斯亮蓝法定量。
1.梯度稀释标准品牛血清白蛋白(BSA,2mg/2mL)制作标准曲线:用PBS缓冲液依次稀释BSA,浓度分别为80μg/μL、40μg/μL、20μg/μL等依次稀释2倍至0μg/μL,每次稀释时均涡旋充分混匀。
2.稀释样品:将每组样品蛋白用PBS缓冲液稀释100倍。
3.上样:取125μL梯度稀释的标准品和待测的样品蛋白加入酶标条中,并加入125μL/孔考马斯亮蓝。
4.用酶标仪进行检测,计算测得样品浓度。
5.根据蛋白上样量和样品浓度调整样品溶液体积,添加适当比例的蛋白上样缓冲液,涡旋混匀,在沸水中煮5min,使蛋白变性,变性后放入-20℃保存。
2.4.3电泳
1.装配垂直电泳装置并检漏。
2.配制浓缩胶与分离胶:
表6:浓缩胶与分离胶的配置
12%分离胶 | 浓缩胶 | |
超纯水 | 1.92mL | 3.40mL |
30%Acr | 2.40mL | 0.83mL |
1.5M Tris-HCL(PH8.8) | 1.56mL | |
1.0M Tris-HCL(PH6.8) | 0.63mL | |
10%SDS | 60μL | 50μL |
10%AP | 60μL | 50μL |
TEMED | 2.4μL | 5μL |
3.制胶:用移液枪将分离胶沿玻璃板一侧缓慢匀速的注入玻璃板中间,注入玻璃板约2/3处,加超纯水封胶,静置20min,待分离胶凝固后缓慢倒掉上层超纯水,再按上述步骤注入浓缩胶,并插上梳子,静置20min。
4.上样:将玻璃板从固定架取出转移至电泳槽,倒入电泳缓冲液,梳子垂直拔出后上样。
5.电泳:先90V电泳30min,待溴酚蓝到达浓缩胶与分离胶交界后将电压调整至
120V,电泳至溴酚蓝跑到分离胶底部,停止电泳。
2.4.4转膜与封闭
取出凝胶板,按照Marker位置裁剪目的蛋白所在区域,剪取同样大小的PVDF膜放入甲醇中激活15s,放在凝胶上面,按照顺序组装转膜夹。组装好后放入转膜仪中,倒入转膜缓冲液,在冰浴中转膜。转膜条件为恒电压100V,1.5h。
转膜完后,将PVDF膜泡于脱脂牛奶中,置于摇床,封闭1.5h。
2.4.5一抗杂交与二抗孵育
1.一抗杂交:按照抗体说明书比例并通过预实验验证,用一抗稀释液以1:500稀释,将PVDF膜放入一抗稀释液中置于摇床4℃过夜。
2.洗膜:移除一抗稀释液,用PBST缓冲液在摇床中洗涤6次,每次5min。
3.二抗孵育:按照抗体说明书比例,用PBST缓冲液以1:1000稀释二抗,将PVDF膜放入二抗稀释液中置于摇床孵育1.5h。
4.洗膜:按照上述洗膜步骤重复一次。
2.4.6ELC显影与图像分析
在暗室中取ECL显色液,将A液与B液等体积混合均匀后滴在保鲜膜上,将PVDF膜放入显色液中反应1min,用化学发光扫描仪进行扫膜。图像用Iamge J软件扫描各个蛋白灰度值(ID值),以β-actin为内参,计算出相对表达量:目的蛋白的相对表达量=目的蛋白ID值/内参ID值。
2.5TYR-si RNA对A375细胞酪氨酸酶表达、细胞凋亡和周期的影响
2.5.1实时荧光定量PCR检测TYR m RNA相对表达水平
1.取对数生长期的黑色素瘤A375细胞,根据细胞传代的方法传代接种于六孔板中,将接种的细胞浓度调整为5×105个每孔,于37℃、5%的CO2培养箱中进行常规培养,待细胞贴壁并生长程度约为50%时,进行转染实验。
2.根据转染操作步骤进行转染,转染浓度分为:100pmol、200pmol、300pmol。
3.根据实时荧光定量PCR的步骤检测黑色素瘤A375细胞的TYR m RNA相对表达水平。
2.5.2Annexin V-FITC/P双染法检测细胞凋亡率
1.取对数生长期的黑色素瘤A375细胞,根据细胞的传代方法将细胞传代接种于六孔板中,将接种的细胞浓度调整为5×105个每孔,于37℃、5%的CO2培养箱中进行常规培养,待细胞贴壁并生长程度约为50%时,进行转染实验。
2.根据细胞转染操作步骤进行转染,转染浓度为100pmol、200pmol、300pmol。培养48h后,收集培养液,用预冷的PBS缓冲液洗涤细胞2次,用无EDTA的0.25%胰蛋白酶将其消化。收集细胞置离心管中。
3.以300g,4℃离心5min,得到细胞沉淀,用预冷的PBS缓冲液重悬细胞沉淀,再以300g,4℃离心5min。
4.重复上述实验步骤一次后,收集1-5×105个细胞。
5.加入100μL1×Binding Buffer重悬细胞。过200目细胞筛后将细胞悬液转移置于流式管中。
6.加入5μL Annexin-V-FITC和5μL PI Staining Solution,轻轻混匀。
7.避光、室温反应10min。
8.加入400μL 1×Binding Buffer,轻轻混匀,样品在1h内用流式细胞仪检测。激发波长为488nm。
2.5.3细胞周期检测
1.取对数生长期的黑色素瘤A375细胞,根据细胞的传代方法将细胞传代接种于六孔板中,将接种的细胞浓度调整为5×105个每孔,于37℃、5%的CO2培养箱中进行常规培养,待细胞贴壁并生长程度约为50%时,进行转染实验。
2.根据细胞转染操作步骤进行转染,转染浓度为100pmol、200pmol、300pmol。转染培养48小时后,收集培养液,用预冷的PBS缓冲液洗涤细胞2次,用无EDTA的0.25%胰蛋白酶将其消化。收集细胞置于离心管中。
3. 1000g离心细胞5min,小心吸除上清,可以残留约50μL左右的培养液,以避免吸走细胞。
4.加入1mL预冷的PBS缓冲液,重悬细胞,吹打均匀后转移至1.5m LEP管内,再次以1000g离心细胞5min,小心吸除上清。轻弹EP管底部以适当分散细胞,避免细胞成团。
5.细胞固定:取1mL预冷的70%乙醇加入EP管中,轻轻吹打细胞使其混匀,放置4℃中固定24h。
6. 1000g离心细胞5min,小心吸除上清。加入1mL预冷的PBS缓冲液,重悬细胞,再次1000g离心细胞5min,小心吸除上清。轻弹EP管底部以适当分散细胞,避免细胞成团。
7.配制碘化丙啶染色液:根据下表配制相对应样品数的碘化丙啶染色液。注:配置好的碘化丙啶染色液短时间内于4℃保存,当日配制当日使用。
表7:碘化丙啶染色液的配置
1个样品 | 6个样品 | |
染色缓冲液 | 0.5mL | 3mL |
碘化丙啶染色液(20X) | 25μL | 150μL |
RNase A(50X) | 10μL | 60μL |
Final volume | 0.535mL | 3.21mL |
8.染色:将每管样品中,加入0.5mL上述新配置的染色液,轻轻吹打细胞,使其充分重悬细胞沉淀。将其放入37℃水浴锅,避光温浴30min。随后放置于4℃避光保存,24h内完成流式检测,激发波长为488nm。
2.6MITF-si RNA对A375细胞酪氨酸酶表达、细胞凋亡和周期的影响
2.6.1实时荧光定量PCR检测m RNA相对表达水平
通过参考文献,得到MITF-si RNA。根据参考文献得到MITF引物,用BLAST验证其特异性后,由生工生物工程股份有限公司合成si RNA与引物。
表8:MITF-si RNA序列
表9:MITF的引物序列
MITF-si RNA转染浓度分别为:100pmol、200pmol、300pmol,根据2.5.1.3的实验步骤,检测不同浓度的MITF-si RNA转染进黑色素瘤A375细胞后,细胞中MITF m RNA、TYR mRNA的相对表达水平。
2.6.2Annexin V-FITC/PI双染法检测细胞凋亡率
MITF-si RNA转染浓度为100pmol、200pmol、300pmol,根据2.5.2的实验步骤,检测不同浓度的MITF-si RNA转染进A375细胞后,细胞的凋亡情况。
2.6.3细胞周期检测
MITF-si RNA转染浓度同100pmol、200pmol、300pmol,根据2.5.3的实验步骤,检测不同浓度的MITF-si RNA转染进A375细胞后,细胞周期的情况。
2.7统计学处理
对数据进行分析使用SPSS 19.0软件,所得到的数据用均值±标准差(x±s)表示。采用单因素方差分析,方差齐用LSD检验方法,方差不齐用Tamhane’s T2检验方法。以P<0.05为检验标准,表示差异有统计学意义。
TYR-siRNA、MITF-siRNA对A375细胞的影响如图5和图6所示。抑制效果最好的干扰片段为siRNA2。
实施例3Arg-脂质体包封鱼精蛋白-siRNA复合体制备
1、将siRNA干粉用12000rpm/min减压离心后,每管加入125μL DEPC水,配制成20μM的溶液,再稀释40倍后备用。
2、鱼精蛋白-siRNA复合体制备:取稀释40倍后的siRNA5.0μL与5μL鱼精蛋白(0.0916mg·mL-1)加HEPES缓冲液(20mM,pH=7.40)稀释成50μL,保持鱼精蛋白和siRNA的质量比为15:1,然后涡旋混匀7min,室温孵育30min,使之形成鱼精蛋白-siRNA复合体。
3、载复合体Arg-脂质体制备:吸取2.0mL Arg-磷脂微囊于离心管中,将制备好的鱼精蛋白-siRNA复合体取100μL加入Arg-磷脂微囊中涡旋5min,充分混匀后置于25℃环境下孵育30min,使Arg-磷脂微囊充分包载鱼精蛋白-siRNA复合体,取出后混匀3min,设置超声波细胞粉碎机的功率为90W,冰水浴超声时间7min,超声完毕后,吸取足够的Arg溶液,使体系中Arg浓度为3.0mM,再次涡旋混匀3min,25℃CO2培养箱中放置2h后取出,设置低温冷冻离心机转速为20000r/min,温度为4℃,时间为10min,高速离心完毕后,吸取离心管底部液体5μL,并通过高分辨&双光子共聚焦显微镜(LSCM)观察荧光成像。在显微镜下可观察到载药Arg-磷脂微囊整齐均匀分布。
由于鱼精蛋白与siRNA之间磷酸-戊糖骨架的静电作用,利用涡旋混匀的方法在室温下孵育制备了鱼精蛋白-siRNA复合体。该核酸类药物鱼精蛋白-siRNA复合体,将对皮肤癌、恶行黑色素瘤等肿瘤等具有治疗作用。发现当鱼精蛋白与siRNA的质量比为15:1时,两者能完全复合。
序列表
<110> 河北大学
<120> Arg-脂质体微囊、微囊包封的鱼精蛋白-siRNA复合体及其制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA/RNA
<213> 人工序列(artificial series)
<400> 1
ccuucuucac caugcauuut t 21
<210> 2
<211> 19
<212> DNA/RNA
<213> 人工序列(artificial series)
<400> 2
gaacgaagaa gaagattta 19
Claims (10)
1.一种Arg-脂质体微囊,其特征是,以磷脂酰胆碱和磷脂酰丝氨酸为原料,采用薄膜分散法制备脂质体微囊,然后在脂质体微囊悬浮液中加入精氨酸,即得所述Arg-脂质体微囊。
2.根据权利要求1所述的Arg-脂质体微囊,其特征是,所述磷脂酰胆碱与磷脂酰丝氨酸的质量比为3-5∶1;精氨酸与脂质体微囊的质量比为0.72-0.73:1。
3.一种权利要求1或2所述的Arg-脂质体微囊在药物载体中的应用。
4.根据权利要求3所述的应用,其特征是,将所述Arg-脂质体微囊作为药物相转变递送载体,Arg-脂质体微囊的相转变温度为41-43℃。
5.一种权利要求1或2所述的Arg-脂质体微囊的制备方法,其特征是,包括以下步骤:
a、将磷脂酰胆碱和磷脂酰丝氨酸溶于氯仿中,其中,磷脂酰胆碱与磷脂酰丝氨酸的质量比为3-5∶1,充分混合均匀后,用氮气吹干氯仿,从而形成薄膜状的脂质体;
b、将所得薄膜状的脂质体置于真空干燥器中干燥0.5-2小时,干燥完全后,加入4-羟乙基哌嗪乙磺酸缓冲液,使脂质体分散悬浮,室温静置0.5-2小时;
c、在脂质体悬浮液中加入精氨酸,其中,精氨酸与脂质体微囊的质量比为0.72-0.73:1,混合均匀后,在冰浴条件下,用细胞破碎仪超声分散,即得所述Arg-脂质体微囊。
6.根据权利要求5所述的制备方法,其特征是,步骤b中,所述4-羟乙基哌嗪乙磺酸缓冲液的浓度为20mM,pH=7.4;步骤c中,超声功率为80-100W,超声时间为5-10min。
7.一种微囊包封的鱼精蛋白-siRNA复合体,其特征是,将鱼精蛋白-siRNA复合体包封于权利要求1或2所述的Arg-脂质体微囊中,所述siRNA为针对酪氨酸酶基因的TYR-siRNA或针对黑色素合成相关MITF基因的MITF-siRNA,所述TYR-siRNA的序列如SEQ:ID:NO:1所示,所述MITF-siRNA的序列如SEQ:ID:NO:2所示。
8.权利要求7所述的微囊包封的鱼精蛋白-siRNA复合体的制备方法,其特征是,包括以下步骤:
a、将siRNA溶液与鱼精蛋白溶液加入到4-羟乙基哌嗪乙磺酸缓冲液中,充分混合均匀后,室温孵育20-40 min,从而得到鱼精蛋白-siRNA 复合体;其中,鱼精蛋白与siRNA的质量比为12-18∶1;
b、将制备好的鱼精蛋白-siRNA 复合体与Arg-脂质体微囊混合,经超声分散后,即得所述微囊包封的鱼精蛋白-siRNA复合体。
9.根据权利要求8所述的制备方法,其特征是,步骤a中,所述4-羟乙基哌嗪乙磺酸缓冲液的浓度为20mM,pH=7.4;步骤b中,超声功率为80-100W,超声时间为5-10min。
10.权利要求7所述的微囊包封的鱼精蛋白-siRNA复合体在护肤品喷剂、面膜或医用敷料中的应用。
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