CN112375785B - 一种促进间充质干细胞成骨分化的方法及应用 - Google Patents

一种促进间充质干细胞成骨分化的方法及应用 Download PDF

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CN112375785B
CN112375785B CN202011291573.6A CN202011291573A CN112375785B CN 112375785 B CN112375785 B CN 112375785B CN 202011291573 A CN202011291573 A CN 202011291573A CN 112375785 B CN112375785 B CN 112375785B
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osteoporosis
nat10
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沈慧勇
杨文�
马梦君
李鸿宇
王鹏
吴燕峰
米汝佳
卢艺萱
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Eighth Affiliated Hospital of Sun Yat Sen University
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Abstract

本发明属于骨组织工程技术领域，具体涉及一种促进间充质干细胞成骨分化的方法及应用，本发明提供了一种促进间充质干细胞成骨分化的方法，即通过生物学方法在体内过表达N‑乙酰基转移酶10(NAT10)，根据该方法通过腺病毒在小鼠体内过表达NAT10，发现NAT10可以逆转骨质疏松小鼠的骨量丢失，增强MSC的成骨分化潜力，有效增加局部骨量，提高骨密度，增强骨力强度，改善局部骨质疏松情况，改善骨代谢，提示本发明可用于构建新型干细胞成骨材料；可见，本发明方法可以为骨质疏松治疗提供新的治疗方案。

Description

一种促进间充质干细胞成骨分化的方法及应用
技术领域
本发明属于骨组织工程技术领域，具体涉及一种促进间充质干细胞成骨分化的方法及应用。
背景技术
骨质疏松症(osteoporosis，简称OP)是一种系统性的代谢性骨病，是因为骨吸收引起骨量的下降，骨质微结构遭到破坏，导致骨质脆性增加而发生的，以易发性骨折为主要特征的全身性骨代谢病(WHO)。同时，骨质疏松也是造成颌骨骨质丢失的主要危险因素之一，可导致牙龈萎缩、牙根暴露，严重者可导致基牙松动脱落，牙槽骨吸收、颌骨萎缩等一系列口腔问题发生，这给临床口腔正畸、义齿修复及种植修复带来了极大的困难。目前此疾病在全球发病率高，危害大，据统计，全球已有累计超过2亿人口患有骨质疏松症，随着年龄的增加，骨质疏松症的患病率正逐渐上升，其发病率已跃居常见疾病的第七位，由于此病患病率较高，严重情况下可对机体造成全身的伤害，WHO已将其列入中老年三大疾病之一，并列为全球公共健康疾病。随着人类寿命延长所带来的老龄化社会的到来，骨质疏松症已成为影响人类健康的重要问题。
目前,骨质疏松症的治疗方式包括调节生活方式，骨健康基本补充剂，抗骨吸收药物和促骨形成药物等，若外伤导致骨折需手术治疗。从青少年期加强运动，保证足够的钙质摄入，同时预防各种疾病可有效减少骨质疏松发生的风险。其中，发生骨质疏松骨折是目前治疗骨质疏松的棘手点，因为骨质疏松骨折的治疗存在骨折愈合时间缓慢，再发生率高等问题。而且如果患者存在高龄，全身健康状况衰退，并存多系统疾病，免疫力低下等情况，手术治疗发生并发症的几率会更高，从而增加了治疗的复杂性与风险性。
因此，寻找新的骨质疏松治疗靶点，研发治疗骨质疏松及其并发症的新型治疗方案具有很重要的应用价值。
发明内容
为了克服上述现有技术的不足，本发明提出一种促进间充质干细胞成骨分化的方法，该方法可以促进成骨分化，增加成骨细胞的数量，改善骨代谢，并且可用于构建新型干细胞成骨材料，为骨质疏松及其并发症治疗提供新的治疗方案。
为了实现上述目的，本发明所采用的技术方案是：
本发明提供了一种促进间充质干细胞成骨分化的方法，即通过生物学方法在体内过表达N-乙酰基转移酶10。
优选地，先制备过表达N-乙酰基转移酶10的表达载体，然后将表达载体导入体内。
优选地，所述表达载体包括但不限于腺病毒和慢病毒。
在人骨质疏松和小鼠骨质疏松模型的股骨成骨细胞表达过程中，N-乙酰基转移酶10(N-acetyltransferase 10,NAT10)降低。在间充质干细胞(MSC)体外成骨过程中，NAT10表达上调，通过将NAT10过表达慢病毒体外转染人MSC，发现MSC成骨分化潜力增强。为此，本发明通过腺病毒在小鼠体内过表达NAT10，发现NAT10可以逆转骨质疏松小鼠的骨量丢失，提示NAT10可用于防治骨质疏松，为骨质疏松及其并发症治疗提供新的治疗方案。
优选地，将表达载体导入体内的方法包括但不限于肌肉注射、皮下注射。
本发明还提供了上述的一种促进间充质干细胞成骨分化的方法在构建用于治疗骨质疏松骨折的新型材料中的应用。
本发明还提供了一种用于治疗骨质疏松的新型材料，将过表达N-乙酰基转移酶10的表达载体与骨诱导磷酸钙生物陶瓷支架经共孵化后得到。
优选地，先将过表达N-乙酰基转移酶10的表达载体导入间充质干细胞诱导培养一段时间后再与骨诱导磷酸钙生物陶瓷支架经进行共孵化。
进一步地，诱导培养的时间不少于7天。
优选地，所述骨诱导磷酸钙陶瓷支架载体为羟基磷灰石/β-磷酸三钙支架(HA/TCP支架)。
根据本发明的促进间充质干细胞成骨分化的方法，本发明将NAT10慢病毒过表达的MSC构建在羟基磷灰石(hydroxyapatite，HA)/β-磷酸三钙(β-tricalcium phosphate，TCP)支架上，构建了一种新型干细胞成骨材料，在裸鼠体内表达可看到裸鼠皮下成骨能力增强，提示本发明方法可为骨质疏松骨折外科治疗提供新型材料。
进一步地，所述羟基磷灰石和β-磷酸三钙的质量比为(2-4):(5-9)。具体地，所述羟基磷灰石和β-磷酸三钙的质量比为3:7。
与现有技术相比，本发明的有益效果是：
本发明提供了一种促进间充质干细胞成骨分化的方法，即通过生物学方法在体内过表达N-乙酰基转移酶10(NAT10)，根据该方法通过腺病毒在小鼠体内过表达NAT10，发现NAT10可以逆转骨质疏松小鼠的骨量丢失，增强MSC的成骨分化潜力，有效增加局部骨量，提高骨密度，增强骨力强度，改善局部骨质疏松情况，改善骨代谢；同时，跟据上述方法，本发明还构建了一种治疗骨质疏松骨折的新型材料，即将NAT10慢病毒过表达的MSC构建在羟基磷灰石(hydroxyapatite，HA)/β-磷酸三钙(β-tricalcium phosphate，TCP)支架上，形成一种新型干细胞成骨材料，在裸鼠体内表达可在裸鼠体内皮下成骨。可见，本发明方法可以为骨质疏松治疗提供新的治疗方案，为骨质疏松骨折外科治疗提供新型材料。
附图说明
图1为动物实验流程图；
图2为模型小鼠的micro-CT成像图；
图3为模型小鼠的骨量参数统计图；
图3中，BV(mm3)：骨体积，指感兴趣区域中被定义为骨组织的体积；TV(mm3)：组织体积，指感兴趣区域总体积，该指标主要根据研究者或研究重点观察区域而定；BV/TV(％)：骨体积分数，表示骨组织体积与组织体积比值，可直接反应骨量变化情况；Tb.N(/mm)：骨小梁数目；Tb.Th(mm)：骨小梁厚度，表示骨小梁平均厚度；Tb.Sp(mm)：骨小梁分离度，表示骨小梁之间髓腔平均宽度；BSA/BV(per mm)：特定骨表面积。
图4为模型小鼠的HE染色图；
图5为裸鼠的HE染色图及Masson三色染色图。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中的实验方法，如无特殊说明，均为常规方法，下述实施例中所用的试验材料，如无特殊说明，均为可通过常规的商业途径购买得到的。
实施例1动物实验：过表达N-乙酰基转移酶10(N-acetyltransferase 10,NAT10)对骨量生成情况的影响
动物实验的流程如图1所示：
(1)动物模型选择及分组
选择8周龄的雌性C57BL/6小鼠，随机分为4组，每组6只。
(2)骨质疏松小鼠动物模型构建
对其中两组8周龄的雌性C57BL/6小鼠实行双侧卵巢切除术(OVX)，分别命名为OVX-NC(卵巢切除正常对照组)和OVX-NAT10 OE(卵巢切除NAT10过表达组)，另外两组实行切口术(假手术)作为空白对照(Sham)，分别命名为Sham-NC(假手术正常对照组)和Sham-NAT10OE(假手术NAT10过表达组)。
(2)过表达NAT10腺病毒的构建
将目的穿梭质粒(pADV-mCMV-Nat10-3xFLAG-P2A-EGFP)或(pADV-mCMV-3xFLAG-P2A-EGFP)与腺病毒骨架质粒(购自和元生物科技有限公司)共转染到HEK293细胞中重组获得病毒，经大量扩增后纯化，检测滴度和目的基因表达水平，构建得到过表达NAT10腺病毒。具体构建方法如下：
转染前一天，将HEK293种到6孔板中，控制细胞转染时的密度为70-80％，转染前1小时，将培养基换成1.5mL Opti-MEM培养基，将待转染的病毒载体质粒4μg(骨架质粒：穿梭质粒＝1:1)溶于Opti-MEM培养基，总体积为250μL，轻轻混匀；将转染试剂稀释液滴加到质粒稀释液中，边加边轻轻混匀后在室温放置20min，使DNA和转染试剂充分结合形成稳定的转染复合体；将上面配好的DNA-转染试剂复合体加入到细胞培养板中，做好标记，放回培养箱；6h后吸去培养基，PBS洗一次，加入2mL新鲜的完全培养基培养；每三天换液一次，大概7-15天左右出现病毒空斑，待完全病变后收集上清液。将HEK293细胞铺于30-40个10cm dish，待细胞长至70-80％，每块板加入合适滴度的病毒(约107-108PFU/mL)10μL，感染细胞,待细胞全部病变后(2-3天)，每块板中加入约500μL 10％Nonidet P40(NP40)以裂解细胞。收集细胞裂解物，12000rpm离心10min，弃细胞碎片收集上清。每100mL上清加入50mL病毒沉淀液(20％PEG8000，2.5M Nacl)，冰上放置1h以沉淀病毒。12000rpm离心上述混合物20min，弃上清，将沉淀物悬浮在10mL密度为1.10g/mL的CsCl溶液中(溶剂为20mM Tris-HCl，pH 8.0)，4℃、7000rpm离心5min，收集病毒悬浮液。在Beckman超速离心管中加入2.0mL的1.40g/mLCsCl溶液(溶剂同上)。再加入3.0mL 1.30g/mL的CsCl溶液。最后加入5mL病毒悬浮液。22800rpm、4℃离心2.5h。收集密度在1.30-1.40g/mL之间的病毒条带至透析袋中(透析袋使用前用10mM的EDTA-Na2煮沸10min)。在透析缓冲液(50g蔗糖，10ml 1M pH为8.0的Tris-HCl液，2ml 1M MgCl2溶液定容至1000ml)中4℃透析过夜，中间更换透析液一次。收集病毒，于-80℃保存。最后构建得到过表达NAT10腺病毒(pADV-mCMV-Nat10-3xFLAG-P2A-EGFP)，并参照上述方法构建得到对照腺病毒(pADV-mCMV-3xFLAG-P2A-EGFP)。
(3)分组注射腺病毒
将上述构建得到的对照腺病毒和过表达NAT10腺病毒分别通过肌肉注射的方式注射在空白对照组或卵巢切除组小鼠的大腿外侧，每两周注射一次，每次注射1x1010[PFU]/mL。
(4)检测小骨量情况
8周后，采用过量麻醉法处死四组小鼠，并取出股骨进行micro-CT扫描，检测并评估骨量生成情况；然后相继进行固定、脱钙、石蜡包埋以及切片处理，通过HE染色检测小鼠骨量的形成情况。
(5)结果
图2的micro-CT测量显示NAT10腺病毒过表达的小鼠，股骨骨小梁明显多于OVX小鼠；图3的骨量参数显示NAT10腺病毒过表达小鼠的BV/TV、Tb.N、Tb.Th较OVX小鼠明显增强，Tb.Sp、BSA/BV较OVX小鼠减弱，说明体内过表达NAT10可以逆转骨质疏松小鼠的骨量丢失；图4的HE染色显示NAT10腺病毒小鼠股骨骨小梁明显比OVX小鼠多，说明体内过表达NAT10的骨质疏松小鼠骨量生成变多，但是NAT10不改变正常小鼠的骨量。
实施例2动物实验：裸鼠皮下的支架成骨情况
(1)动物模型选择及分组
选择8周龄的雌性BALB/c裸鼠，随机分为2组，每组6只。
(2)NAT10过表达慢病毒载体的构建
转染前一天，将293T细胞消化后传代至100mm dish中，控制细胞转染时密度为70～80％；转染前1小时将培养基换成10mL的Opti-MEM培养基；将待转染的病毒载体质粒32ug(骨架质粒：穿梭质粒＝1:1)(骨架质粒为和元生物技术股份有限公司提供，穿梭质粒为Ubi-MSC-3FLAG-SV40-EGFP-IRES-puromycib/Ubi-MSC-NAT10-3FLAG-SV40-EGFP-IRES-puromy cibn)溶于Opti-MEM培养基，总体积为500uL，轻轻混匀，静置5分钟；将转染试剂溶于Ooti-MEM培养基，总体积500uL，轻轻混匀，静置5分钟，使DNA和转染试剂充分结合形成稳定的转染复合物；将上面配好的DNA-转染试剂复合物加入到细胞培养板中；6小时后用PBS洗一次，加入10mL新鲜的完全培养基。转染48小时后，收集培养后的上清至50mL离心管中，并更换新鲜的完全培养基；转染72小时后，再次收集培养基，与48小时收集的上清合并，构建得到NAT10过表达慢病毒载体LV-NAT10(Ubi-MSC-NAT10-3FLAG-SV40-EGFP-IRES-puromycibn)，参照上述方法构建得到对照NC慢病毒载体CON238(Ubi-MSC-3FLAG-SV40-EGFP-IRES-puromycibn)。
(3)成骨材料构建
通过Lipofectamine 3000转染试剂盒(Invitrogen)进行转染，将上述2种病毒载体(1.5μg/孔)转入第4代MSC中，36小时后，用成骨分化诱导培养基[含1000mg/L葡萄糖的细胞培养基DMEM，10％胎牛血清，1％青霉素-链霉素(Gibco)，10mM max-甘油磷酸盐(Sigma-Aldrich)，0.1M地塞米松(MB1434，大连美仑生物技术有限公司)和50M抗坏血酸(Sigma-Aldrich)]诱导培养7天，然后将MSC(5×105个细胞)与40mg骨诱导磷酸钙生物陶瓷支架[HA/TCP支架，四川拜阿蒙生物活性材料公司，规格THL/φ9×2，羟基磷灰石(hydroxyapatite，HA)：β-磷酸三钙(β-tricalcium phosphate，TCP)＝3:7，即12mg的HA和28mg的TCP]于37℃培养箱共孵化1小时，1500rpm转速离心5分钟，得到分别由含NAT10过表达慢病毒和NC慢病毒的MSC所构建的HA/TCP支架。
(4)将成骨材料移植于裸鼠皮下
分别将由含NAT10过表达慢病毒和NC慢病毒的MSC所构建的HA/TCP支架随机皮下注射至两组裸鼠皮下，每次注射1x1010[PFU]/mL。
(5)检测支架成骨情况
8周后，采用颈椎脱臼法处死裸鼠，将支架取出，用4％多聚甲醛固定，并用10％EDTA(pH 7.4)脱钙2周，然后经石蜡包埋并切片(5-μm厚)处理，最后进行HE及Masson三色染色。
(6)结果
在H&E染色中，紫蓝色为细胞核，红色为细胞质和细胞外基质(包括骨基质)。Masson染色中，蓝色为胶原纤维(骨基质的主要有机成分，黑色箭头所示)，黑色为细胞核，红色为细胞质、肌肉纤维和红细胞。
图5的HE及Masson染色显示，NAT10过表达支架，MSC产生的胶原比对照组多，说明NAT10过表达支架较NC支架的成骨分化能力强。
以上对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。

Claims (5)

1.N-乙酰基转移酶10在构建用于治疗骨质疏松的新型材料中的应用，其特征在于，先将过表达N-乙酰基转移酶10的表达载体导入间充质干细胞诱导培养一段时间后再与骨诱导磷酸钙生物陶瓷支架进行共孵化。
2.一种用于治疗骨质疏松的新型材料，其特征在于，先将过表达N-乙酰基转移酶10的表达载体导入间充质干细胞诱导培养一段时间后再与骨诱导磷酸钙生物陶瓷支架进行共孵化。
3.根据权利要求2所述的用于治疗骨质疏松的新型材料，其特征在于，诱导培养的时间不少于7天。
4.根据权利要求2所述的用于治疗骨质疏松的新型材料，其特征在于，所述骨诱导磷酸钙生物陶瓷支架为羟基磷灰石/β-磷酸三钙支架。
5.根据权利要求4所述的用于治疗骨质疏松的新型材料，其特征在于，所述羟基磷灰石和β-磷酸三钙的质量比为(2-4):(5-9)。
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