CN106388967A - 一种BMP‑9基因结合Aln对骨质疏松内固定的模型 - Google Patents
一种BMP‑9基因结合Aln对骨质疏松内固定的模型 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2267/03—Animal model, e.g. for test or diseases
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明公开了一种BMP‑9基因结合Aln对骨质疏松内固定的模型,取6月龄Sprague‑Dawley雌性大鼠64只;12h更替黑夜和白昼,环境温度22℃,自由摄食饮水,喂养啮齿类动物标准饲料,每笼4只,适应性喂养7d后进行动物实验;制作骨质疏松大鼠模型后按完全随机区组设计分为:单纯螺钉固定组、Aln组、BMP‑9转染组、BMP‑9转染+Aln组,每组16只;在螺钉植入后4w、8w每组各处死8只大鼠,进行实验指标检测。本发明BMP‑9基因结合Aln能显著提高螺钉周围的骨量,提高螺钉的抗拔出能力,提高骨与螺钉的接触率;在增加金属内植物周围骨组织量、增加与骨的接触方面具有交互作用。
Description
技术领域
本发明属于骨质疏松内固定技术领域,尤其涉及一种BMP-9基因结合阿伦磷酸钠(Alendronate Aln)对骨质疏松内固定的模型。
背景技术
骨质疏松时,患者的骨代谢处于高转换状态,骨的生物机械强度减低,骨脆性增加,在需要行植入物内固定手术时,钻孔或螺钉、假体的植入容易引起细微骨折和局部热损伤,诱发早期内植物周围的炎性反应,使破骨细胞聚集增多,引起骨溶解或骨吸收发生,而骨质疏松患者体内自身修复的因子表达缺乏,骨修复作用降低,最终导致螺钉松动,假体的移位、下沉,手术失败。近年来,内植物-骨界面的稳定性研究主要集中在对内植物设计和对内植物表面的修饰处理上,如通过螺钉接骨板的锁定机制提高螺钉骨界面抗拔出性能,减少早期螺钉切割,或通过植入假体表面羟基磷灰石涂层,增加骨长入。虽然可在一定程度上减少内固定的失败,但提高内植物周围骨质量,增强内植物-骨界面固定强度才是最终的解决办法。研究发现,内植物植入骨内后,周围骨组织结构的改建,涉及众多细胞类型参与及不同因子的调节,类似于骨折的修复。而骨折的修复,BMP(bone morphogenetic proteins,BMP)起着关键作用。BMP是早期诱导骨组织形成与发生的重要信号分子,可诱导未分化的间充质干细胞分化为成骨细胞,在维持骨的矿物质含量和骨改建方面起着重要作用。
在目前已知的30多种BMP中,BMP-9诱导成骨作用最强。通过外源性BMP局部给药促进骨再生,具有持续时间短、用量大、反复追加用药、成本高等缺点。为克服此缺点,将转基因技术将目的基因导入种子细胞,使其在植入部位持续表达,达到治疗的目的。阿伦磷酸钠(Alendronate Aln)是临床常用抗骨质疏松药,其抗骨质疏松主要作用机制是抑制破骨细胞前体细胞向骨骼表面游走和募集,并可抑制它们继续向多核细胞分化。同时使成熟的破骨细胞形态改变、功能丧失,并促进破骨细胞的凋亡。阿仑磷酸纳除抑制破骨细胞引起的骨吸收外,对成骨细胞也有促进作用。骨髓间充质干细胞(Bone marrow mesenchymal stemcells,BMSCs)具有来源丰富、易于取材,具有强大的增殖及多细胞分化潜能,并且低免疫原性和免疫调节作用,已广泛作为基因治疗的载体细胞。
目前在BMP-9基因转染结合Aln治疗对骨质疏松状态下金属骨内植物稳定性作用的影响上还没有相关资料进行报道。
发明内容
本发明的目的在于提供一种BMP-9基因结合Aln对增强骨质疏松内固定的模型,旨在评估BMP-9基因转染结合Aln治疗对骨质疏松状态下金属骨内植物稳定性作用的影响问题。
本发明是这样实现的,BMP-9基因结合Aln对骨质疏松内固定的模型为6月龄Sprague-Dawley雌性大鼠64只,体重275±25g,SPF级;管理饲养,12h更替黑夜和白昼,环境温度22℃,自由摄食饮水,喂养啮齿类动物标准饲料,每笼4只,适应性喂养7d后进行动物实验;大鼠按完全随机区组设计分为:单纯螺钉固定组、阿伦磷酸钠组、BMP-9转染组、BMP-9转染+阿伦磷酸钠组,每组16只,在螺钉植入后4w、8w每组各处死8只大鼠,进行实验指标检测。
本发明的另一目的在于提供一种所述BMP-9基因结合Aln对骨质疏松内固定的模型的建立方法,所述建立方法包括以下步骤:
大鼠称重后,用3.5%水合氯醛按1ml/100g体重腹腔内注射麻醉,待麻醉显效后,手术区备皮,取俯卧位,将大鼠四肢固定在手术台上,以碘伏消毒手术区,铺无菌单;
以大鼠背侧骶前横突最长处近端1cm为中心,延正中线作长约1.0cm—1.5cm的纵向皮肤切口,先将皮肤切口向右侧牵拉,显露腹肌在背侧止点,延其止点用眼科剪剪开,于骶脊肌外侧缘可见腹膜内一乳白色发亮脂肪团,用血管钳将脂肪团轻轻夹出切口,在脂肪团内可见一绿豆大小棕黄色卵巢及相连输卵管,有时可见卵巢表面黄色卵泡,用止血钳夹住卵巢,用0号丝线距卵巢约0.5cm处结扎输卵管,切除卵巢,将余下部分回纳入腹腔,缝合肌肉附着点。将皮肤切口牵向左侧,用同样方法切除左侧卵巢;
缝合皮肤,碘伏再次消毒切口。将所切除的卵巢组织用4%的多聚甲醛固定,石蜡包埋切片,HE染色,行显微镜观察,确定所切组织为卵巢。术后放入笼中,常规喂养三个月。
进一步,所述建立方法还包括螺钉植入模型建立方法,具体包括:
3.5%水合氯醛麻醉,显效后,双侧膝内外侧区备皮,将大鼠取仰卧位,四肢固定于自制操作台上,术区碘伏消毒铺巾;
于右侧膝下内侧切开皮肤,长1cm,显露胫骨平台内侧,用尖刀剥离表面骨膜,辨清平台上缘及胫骨前后缘,于距平台上缘3mm,前后缘中点处以Φ1.2mm空针头开口,以Φ1.2mm钻头钻深约4mm,放入明胶海绵,植入Φ1.5mm*4mm钛螺钉,缝合皮肤。
本发明的另一目的在于提供一种所述BMP-9基因结合Aln对骨质疏松内固定的模型的应用,所述应用包括:
利用HEK293细胞扩增获取高滴度的重组腺病毒BMP-9,不同滴度转染大鼠BMSCs,当MOI值为100时,Ad-BMP-9腺病毒转染BMSCs,BMP-9稳定表达,Ad-BMP-9腺病毒转染大鼠BMSCs的转染效率为80%~90%,细胞的增殖不受转染影响;
64只6月龄SD大鼠随机分成4组,每组16只:单纯螺钉固定组、阿伦磷酸钠组、BMP-9转染组、BMP-9转染+阿伦磷酸钠组;双侧卵巢切除后3月制作骨质疏松模型,分别于双侧胫骨近心端植入螺钉,BMP-9组和BMP-9+Aln组同时植入转染有BMP-9的BMSCs;
在螺钉植入后即刻,Aln组和BMP-9+Aln组分别以1ml/kg皮下注射Aln,每周2次;其余组分别皮下注射等量生理盐水。在螺钉植入后4w、8w每组各处死8只大鼠,进行生物力学测定、Micro-CT扫描、硬组织切片V-G染色、脱钙骨BMP-9免疫组化染色。
本发明提供的BMP-9基因结合Aln对骨质疏松内固定的模型,Aln与BMP-9联合使用具有协同促进成骨的作用,并且Aln能抑制BMP-9对破骨细胞调节的正面作用,抑制BMP-9诱导BMSc向破骨样细胞分化。因此,本部分研究主要是通过建立大鼠骨质疏松模型,在体内研究BMP-9结合二磷酸盐治疗对内植物固定作用的影响。4w、8w时BMP-9+Aln组螺钉拔出力比Control组提高1倍多,比单纯BMP-9组和Aln组也有显著升高(P<0.01);Micro-CT结果显示BMP-9+Aln组BV/TV、TB.TH、Tb.N升高分别是Control组的4倍、1倍、2倍左右,但Tb.Sp不到Control组的一半(P<0.01),Aln组和BMP-9组BV/TV、TB.TH、Tb.N也明显增加,但效果较BMP-9+Aln组差,Tb.Sp降低也差于BMP-9+Aln组。硬组织切片V-G染色显示4w时,BMP-9+Aln组螺钉螺纹间可见大量骨组织长入,周围也有较多骨质形成,其螺钉与骨的接触率比Control组多近2倍(P<0.01),Aln组和BMP-9组螺纹间也有部分骨质长入,BMP-9显著多于Aln组(P<0.01),但螺钉周围骨组织量要少于Aln组;8w时,与Control组相比,各组间均有较多骨组织形成,螺纹间骨质长入增多,以BMP-9+Aln组最明显,其螺钉与骨的接触率比Control组多近1倍(P<0.01),BMP-9螺纹间骨长入增多,骨接触率明显高于Aln组,但周围骨组织少于Aln组。BMP-9免疫组化染色显示4w时BMP-9组和BMP-9+Aln组可间成骨细胞和成熟骨细胞的胞浆内BMP-9阳性表达,8w时表达减弱,而Control组和Aln组一直未见BMP-9明显阳性表达。
转染有BMP-9基因的BMSCs细胞植入体内后,可在骨形成过程中持续表达BMP-9,Aln能显著提高螺钉周围的骨量,提高螺钉的抗拔出能力,相对于Aln,BMP-9改善螺钉固定主要是通过提高螺纹间的骨长入,提高骨与螺钉的接触率,当BMP-9基因转染结合Aln治疗时能显著提高骨质疏松症内植物在骨内的稳定,在增加内植物周围骨组织量,增加与骨的接触方面具有交互作用。
附图说明
图1是本发明实施例提供的BMP-9基因结合Aln对骨质疏松内固定的模型流程图。
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。
下面结合附图对本发明的应用原理作详细的描述。
如图1所示,本发明实施例的BMP-9基因结合Aln对骨质疏松内固定的模型的构建方法包括以下步骤:
S101:大鼠称重后,用3.5%水合氯醛按1ml/100g体重腹腔内注射麻醉,待麻醉显效后,手术区备皮,取俯卧位,将大鼠四肢固定在手术台上,以碘伏消毒手术区,铺无菌单;
S102:以大鼠背侧骶前横突最长处近端1cm为中心,延正中线作长约1.0cm—1.5cm的纵向皮肤切口,先将皮肤切口向右侧牵拉,显露腹肌在背侧止点,延其止点用眼科剪剪开,于骶脊肌外侧缘可见腹膜内一乳白色发亮脂肪团,用血管钳将脂肪团轻轻夹出切口,在脂肪团内可见一绿豆大小棕黄色卵巢及相连输卵管,有时可见卵巢表面黄色卵泡,用止血钳夹住卵巢,用0号丝线距卵巢约0.5cm处结扎输卵管,切除卵巢,将余下部分回纳入腹腔,缝合肌肉附着点。将皮肤切口牵向左侧,用同样方法切除左侧卵巢;
S103:缝合皮肤,碘伏再次消毒切口。将所切除的卵巢组织用4%的多聚甲醛固定,石蜡包埋切片,HE染色,行显微镜观察,确定所切组织为卵巢。术后放入笼中,常规喂养三个月。
下面结合实验对本发明的应用原理作进一步的描述。
1材料与方法
1.1材料
1.1.1主要实验仪器
其它耗材及仪器同实验一、实验二
及实验三
1.1.2主要试剂配置
(1)骨标本浸液I:甲基丙烯酸甲酯75ml、邻苯二甲酸二丁酯25ml,磁力搅拌混匀3h,常温保存备用。
(2)骨标本浸液II:甲基丙烯酸甲酯75ml、邻苯二甲酸二丁酯25ml、过氧化苯甲酰1g,磁力搅拌混匀4h,常温保存备用。
(3)骨标本浸液III:甲基丙烯酸甲酯75ml、邻苯二甲酸二丁酯25ml、过氧化苯甲酰2.5g,搅拌混匀6h,常温保存备用。
(4)10%的EDTA溶液配制:100gEDTA溶于磷酸盐缓冲液(PBS)1000ml中,用HCL调节PH为PH6.4-6.8,高温消毒后常温放置备用。
1.1.3实验动物及分组
6月龄Sprague-Dawley(SD)雌性大鼠64只,体重275±25g,SPF级,购自重庆医科大学动物实验中心,动物许可证号:CQLA—2012-0512。大鼠由重庆医科大学动物实验中心(SPF实验室)管理饲养,12h更替黑夜和白昼,环境温度22℃,自由摄食饮水,喂养啮齿类动物标准饲料,每笼4只,适应性喂养7d后进行动物实验。
大鼠按完全随机区组设计分为:单纯螺钉固定组(Control组)、阿伦磷酸钠组(Aln组)、BMP-9转染组(BMP-9组)、BMP-9转染+阿伦磷酸钠组(BMP-9+Aln组),每组16只。在螺钉植入后4w、8w每组各处死8只大鼠,进行实验指标检测。
1.2方法
1.2.1骨质疏松大鼠动物模型建立
①大鼠称重后,用3.5%水合氯醛按1ml/100g体重腹腔内注射麻醉,待麻醉显效后,手术区备皮,取俯卧位,将大鼠四肢固定在自制手术台上,以碘伏消毒手术区,铺无菌单;
②以大鼠背侧骶前横突最长处近端约1cm为中心,延正中线作长约1.0cm—1.5cm的纵向皮肤切口,先将皮肤切口向右侧牵拉,显露腹肌在背侧止点,延其止点用眼科剪剪开,于骶脊肌外侧缘可见腹膜内一乳白色发亮脂肪团,用血管钳将脂肪团轻轻夹出切口,在脂肪团内可见一绿豆大小棕黄色卵巢及相连输卵管),有时可见卵巢表面黄色卵泡,用止血钳夹住卵巢,用0号丝线距卵巢约0.5cm处结扎输卵管,切除卵巢,将余下部分回纳入腹腔,缝合肌肉附着点。将皮肤切口牵向左侧,用同样方法切除左侧卵巢。
③缝合皮肤,碘伏再次消毒切口。将所切除的卵巢组织用4%的多聚甲醛固定,石蜡包埋切片,HE染色,行显微镜观察,确定所切组织为卵巢。术后放入笼中,常规喂养三个月。
1.2.2骨密度(Bone mineral density,BMD)测量
术前和术后3个月,随机从行双侧卵巢切除的大鼠中抽取12只,经用3.5%水合氯醛按1ml/100g麻醉后,四肢展开,俯卧于双能X线吸收测量仪平台上,通过小动物软件测量量腰4、5椎体BMD值,采用配对t检验对其BMD值进行分析。
1.2.3MSCs与可吸收明胶海绵复合培养
利用HEK293细胞扩增获取高滴度的重组腺病毒BMP-9,不同滴度转染大鼠BMSCs,当MOI值为100时,Ad-BMP-9腺病毒转染BMSCs,BMP-9稳定表达,Ad-BMP-9腺病毒转染大鼠BMSCs的转染效率为80%~90%,MTT法测定细胞生长曲线,细胞的增殖不受转染影响,细胞的增殖不受转染影响,符合实验要求。
Ad-BMP9转染细胞及未转染的细胞于转染后72h,0.25%胰酶消化收集,每块可吸收明胶海绵按1×106个BMSc接种。为使细胞尽量多地进入材料内部,对可吸收明胶海绵进行预处理:将明胶海绵在无菌条件下剪成3mm×3mm大小块状,加入含20%胎牛血清的L-DMEM完全培养液置入37℃、5%CO2饱和湿度孵箱中过夜后于荧光显微镜下观察。手术时,Control组和Aln组植入培养有BMSc的可吸收明胶海绵,BMP-9组和BMP-9+Aln组植入培养有转染BMP-9细胞的可吸收明胶海绵。
1.2.4螺钉植入模型建立
钛螺钉及手术器械均于术前一日高温高压消毒灭菌,手术遵循手术无菌操作原则。
①3.5%水合氯醛(1ml/100g)麻醉,显效后,双侧膝内外侧区备皮,将大鼠取仰卧位,四肢固定于自制操作台上,术区碘伏消毒铺巾;
②于右侧膝下内侧切开皮肤,长约1cm,显露胫骨平台内侧,用尖刀剥离表面骨膜,辨清平台上缘及胫骨前后缘,于距平台上缘3mm,前后缘中点处以Φ1.2mm空针头开口,以Φ1.2mm钻头钻深约4mm,放入明胶海绵(大小为2mm×2mm,利用取出钻头的负压将明胶海绵吸入),植入Φ1.5mm*4mm钛螺钉,缝合皮肤;
③手术操作左侧同右侧;
1.2.5给药方式
将Aln纯粉由生理盐水配成50μg/ml混悬液备用,用前摇匀。在螺钉植入后即刻,Aln组和BMP-9+Aln组以1ml/kg皮下注射Aln,每周2次;其余组分别皮下注射等量生理盐水。
1.2.6生物力学测定
螺钉植入后4w、8w,每组各处死8只大鼠,取植入钛螺钉的左侧胫骨,采用BOSE3330动态疲劳试验机,将骨端置于试验机下端夹具的夹曹内,螺钉帽部分植入上端自制夹具内,初始载荷为0.1N,以1N/0.01s速度递增,扫描间隔为0.001s,当螺钉完全拔出后停止受力。
1.2.7Micro-CT扫描
螺钉植入后4w、8w,每组各处死8只大鼠,取右侧胫骨,以4%的多聚甲醛固定。为避免金属螺钉对扫描结果的影响,MicroCT扫描前,小心取出钛螺钉,将样本置于Micro-CT系统的检测试管内,垂直于标本长轴方向扫描,图像分辨率2048×2048,素点为20×20μm,层间距20μm。电压55KV,145mA,整合时间为380ms扫描完成后用Microview V2.1.2分析软件进行三维重建分析。CT值高于270定为骨组织,设出兴趣区域(range of interests,ROI)为从皮质骨下20层开始,以螺钉为中心,半径为2.5mm的圆面积,连续扫描80层,检验参数包括:(1)骨体积分数(bone volume fraction,BVF):骨小梁的体积(bone volume,BV)/样本的体积(total volume,TV)与,即BV/TV;(2)骨小梁间隙(trabecular spacing,Tb.Sp);(3)骨小梁厚度(trabecular thickness,Tb.Th);(4)骨小梁数目(trabecular number,Tb.N)
1.2.8硬组织切片观察
在行Micro-CT扫描后,将4枚标本进行硬组织切片,观察骨接触情况。
硬组织切片制备
(1)骨组织包埋方法;
①固定和脱水:待4%多聚甲醛中固定2天后,分别于70%乙醇—95%乙醇—100%乙醇行梯度脱水,依次各1d,脱水后用二甲苯透明脱脂8h。
②浸透:骨标本按浸液I—浸液II—漫液III的顺序,依次各浸透2d。
③包埋:将已浸透的标本放入15ml大小的玻璃瓶内,倒入现配的浸液III中(每个标本10m1),瓶盖盖好后插上针头排气,置入4℃冰箱中5d,然后放于室温下继续固化。
④固化后,将其放于40℃烘箱内直至完全硬化(用针头刺其表面,不能刺入为准),切片前砸碎玻璃。
(2)骨标本切片:
于修块机上将包块修整,使之能牢固固定在切片机上,且使刀片切割方向与螺钉长轴方向垂直,即延失状面切割,切片厚度约为150μm。切割后超声清洗切片10s,ddH2O冲洗2min,常温晾干,用粘合剂将切片粘合至硬组织切片专用有机玻片上,并使用5kg砝码压片24h。
手工膜片:先在磨砂玻璃上膜片,然后分别用P300、P800、P1200砂纸磨片,将切片磨成厚度约50μm,再对磨片进行抛光,最终切片厚度约30~40μm
(3)V-G染色法(VAN GIESON染色—苦味酸品红染色)
①超声洗片2min,ddH2O冲洗2min;
②将切片放于0.1%甲酸3min后,用流水冲洗2min;
③将切片放于20%甲醇2min,流水冲洗2min;
④在60℃恒温箱中用Stevenol蓝染色15min后,流水冲洗2min,并擦干;
⑤室温下将切片进行V-G染染色8min,水洗后晾干与显微镜下观察。
(4)新生骨接触情况观察
用Olympus光学显微镜(BX51,Olympus,Jappan)观察切片,并用Image-proplus(xmage-PrPlusR,Mediaeyberneties,silver springs,Mn,usA)自动图像处理软件进行骨组织形态计量学的测量。
螺钉-骨接触率=(螺钉与骨接触的长度/螺钉在骨内的总长度)X100%
1.2.9免疫组化染色
在行Micro-CT扫描后,将4枚标本进行脱钙,用于BMP-9免疫组化染色。
(1)骨组织脱钙:
①将标本固定24~48h后,用PBS冲洗3次,每次20min;
②将标本移植10%EDTA脱钙液中,每标本脱钙液约10ml,常温下保存;
③每日摇动脱钙液,让标本与脱钙用充分接触,4~5d时更换脱钙液;
④4w~5w时用针头刺骨组织,如能轻松刺入说明脱钙成功;
⑤脱钙成功后,H2O冲洗60min,进行石蜡包埋切片。
(2)免疫组化染色
①石蜡切片脱蜡脱水:将标本放入二甲苯I、II中各10min。
②梯度酒精脱水:100%酒精,2min—95%酒精,2min—80%酒精,2min—70%酒精,2min。
③脱水后,用dH2O洗2次,各5min;
④用过氧化氢避光封闭内源性过氧化物酶10min;
⑤dH2O洗2次各5min;
⑥抗原修复:将标本置于塑料切片架,放入耐温玻璃容器内,用构椽酸钠缓冲液淹没切片,放入微波炉内,选择中高档,5min;取出并补充已预热的构椽酸钠缓冲液;再选择中高档,5min。
⑦PBS冲洗2次,各5min。
⑧血清封闭:擦净切片周围的水份(保持组织呈湿润状态),滴加二抗血清,37℃孵育30min。
⑨滴加一抗:用滤纸吸去血清,滴加一抗,37℃孵育2h。
⑩PBS小心冲洗2次,每次5min。
滴加生物素化的二抗,37℃,40min。
用PBS冲洗2次,每次5min。
滴加三抗(SAB复合物),37℃,40min。
再用PBS冲洗2次,每次5min后看,用DAB显色,镜下观察,适时自来水冲洗终止。
苏木素复染,于室温下放置30s后自来水冲洗;
梯度酒精脱水后,用二甲苯透明:I,11(二甲苯)各5min
用树胶封片后镜下观察。
1.3统计学分析
数据采用SPSS13.0统计软件进行分析,结果以均数±标准差表示,组间比较采用单因素方差分析,组内的两两比较采用多个样本q检验,检验标准为a=0.05,P﹤0.05为差异有统计学意义。
2结果
2.1大鼠腰椎BMD测量
大鼠卵巢切除术(OVX)术前BMD结果为1.366±0.043g/cm2,OVX术后3月BMD均值低于术前均值的-3SD。按照WHO骨质疏松诊断标准中T<-2.5,ovx术后3月满足骨质疏松诊断标准,术后3月腰椎BMD值为1.056±0.049g/cm2。
2.2生物力学实验
4W时Aln组和BMP-9组螺钉拔出力均较Control组明显升高(P<0.01),但Aln组升高较BMP-9组明显(P<0.01),到8W时,Aln组拔出力仍高于BMP-9组,但统计学差异不显著(P>0.01)。4W、8W时BMP-9+Aln组螺钉拔出力均明显高于单独Aln和BMP-9组(P<0.01)。
2.3Micro-CT结果
Micro-CT结果显示,4w、8w时联合应用Aln和BMP-9,BV/TV增高是Control组的4倍多(P<0.01),Aln组和BMP-9组也明显增加(P>0.01),与单独使用BMP-9、Aln相比,联合运用升高明显(P<0.01)。
4w时,Aln组和BMP-9+Aln组TB.TH升高是Control组的1倍(P<0.01),到第8w时,也有明显升高(P<0.01),但BMP-9+Aln组好于Aln组。BMP-9组比Control组升高20%~30%,但统计学差异不显著(P>0.01)。
4w、8w时Tb.N结果显示BMP-9+Aln组比Control组增高近2倍(P<0.01),单独使用BMP-9、Aln也有升高(P<0.01),但低于BMP-9+Aln组。
4w、8w时结果显示BMP-9+Aln组Tb.Sp不到Control组的一半(P<0.01),Aln组和BMP-9组也有降低,但少于BMP-9+Aln组
2.4骨与螺钉接触情况
4w时,BMP-9+Aln组螺钉螺纹间可见大量骨组织长入,周围也有较多骨质形成,其螺钉与骨的接触率比Control组多近2倍(P<0.01),Aln组和BMP-9组螺纹间也有部分骨质长入,BMP-9显著多于Aln组(P<0.01),但螺钉周围骨组织量要少于Aln组。
8w时,与Control组相比,各组间均有较多骨组织形成,螺纹间骨质长入增多,以BMP-9+Aln组组最明显,其螺钉与骨的接触率比Control组多近1倍(P<0.01),BMP-9螺纹间骨长入增多,骨接触率明显高于Aln组,但周围骨组织少于Aln组。
2.5免疫组化染色结果
免疫组化结果显示,4w时BMP-9组和BMP-9+Aln组可间成骨细胞和成熟骨细胞的胞浆内BMP-9阳性表达,8w时表达减弱,而Control组和Aln组一直未见BMP-9明显阳性表达。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,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,均应包含在本发明的保护范围之内。
Claims (4)
1.一种BMP-9基因结合Aln对骨质疏松内固定的模型,其特征在于,所述BMP-9基因结合Aln对骨质疏松内固定的模型为:6月龄Sprague-Dawley雌性大鼠64只,体重275±25g,SPF级;管理饲养,12h更替黑夜和白昼,环境温度22℃,自由摄食饮水,喂养啮齿类动物标准饲料,每笼4只,适应性喂养7d后进行动物实验;大鼠按完全随机区组设计分为:单纯螺钉固定组、阿伦磷酸钠组、BMP-9转染组、BMP-9转染+阿伦磷酸钠组,每组16只;在螺钉植入后4w、8w每组各处死8只大鼠,进行实验指标检测。
2.一种如权利要求1所述BMP-9基因结合Aln对骨质疏松内固定的模型的建立方法,其特征在于,所述建立方法包括以下步骤:
大鼠称重后,用3.5%水合氯醛按1ml/100g体重腹腔内注射麻醉,待麻醉显效后,手术区备皮,取俯卧位,将大鼠四肢固定在手术台上,以碘伏消毒手术区,铺无菌单;
以大鼠背侧骶前横突最长处近端1cm为中心,延正中线作长约1.0cm—1.5cm的纵向皮肤切口,先将皮肤切口向右侧牵拉,显露腹肌在背侧止点,延其止点用眼科剪剪开,于骶脊肌外侧缘可见腹膜内一乳白色发亮脂肪团,用血管钳将脂肪团轻轻夹出切口,在脂肪团内可见一绿豆大小棕黄色卵巢及相连输卵管,有时可见卵巢表面黄色卵泡,用止血钳夹住卵巢,用0号丝线距卵巢约0.5cm处结扎输卵管,切除卵巢,将余下部分回纳入腹腔,缝合肌肉附着点,将皮肤切口牵向左侧,用同样方法切除左侧卵巢;
缝合皮肤,碘伏再次消毒切口,将所切除的卵巢组织用4%的多聚甲醛固定,石蜡包埋切片,HE染色,行显微镜观察,确定所切组织为卵巢,术后放入笼中,常规喂养三个月,制作骨质疏松大鼠模型。
3.如权利要求2所述的建立方法,其特征在于,所述建立方法还包括螺钉植入模型建立方法,具体包括:
3.5%水合氯醛麻醉,显效后,双侧膝内外侧区备皮,将大鼠取仰卧位,四肢固定于自制操作台上,术区碘伏消毒铺巾;
于右侧膝下内侧切开皮肤,长1cm,显露胫骨平台内侧,用尖刀剥离表面骨膜,辨清平台上缘及胫骨前后缘,于距平台上缘3mm,前后缘中点处以Φ1.2mm空针头开口,以Φ1.2mm钻头钻深约4mm,放入明胶海绵,植入Φ1.5mm*4mm钛螺钉,缝合皮肤。
4.一种如权利要求1所述BMP-9基因结合Aln对骨质疏松内固定的模型的应用,其特征在于,所述应用包括:
利用HEK293细胞扩增获取高滴度的重组腺病毒BMP-9,不同滴度转染大鼠BMSCs,当MOI值为100时,Ad-BMP-9腺病毒转染BMSCs,BMP-9稳定表达,Ad-BMP-9腺病毒转染大鼠BMSCs的转染效率为80%~90%,细胞的增殖不受转染影响;
64只6月龄SD大鼠随机分成4组,每组16只:单纯螺钉固定组、阿伦磷酸钠组、BMP-9转染组、BMP-9转染+阿伦磷酸钠组;双侧卵巢切除后3月制作骨质疏松模型,分别于双侧胫骨近心端植入螺钉,BMP-9组和BMP-9+Aln组同时植入转染有BMP-9的BMSCs;
在螺钉植入后即刻,Aln组和BMP-9+Aln组分别以1ml/kg皮下注射Aln,每周2次;其余组分别皮下注射等量生理盐水,在螺钉植入后4w、8w每组各处死8只大鼠取双侧胫骨标本,进行生物力学测定、Micro-CT扫描、硬组织切片V-G染色、脱钙骨BMP-9免疫组化染色。
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CN111670859A (zh) * | 2020-05-14 | 2020-09-18 | 广州中医药大学第三附属医院(广州中医药大学第三临床医学院、广州中医药大学附属骨伤科医院、广州中医药大学骨伤科研究所) | 一种肌少-骨质疏松症大鼠模型的构建方法 |
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CN111543388A (zh) * | 2019-09-20 | 2020-08-18 | 华中科技大学同济医学院附属协和医院 | 一种去势联合药物建造小鼠骨质疏松模型的方法 |
CN111670859A (zh) * | 2020-05-14 | 2020-09-18 | 广州中医药大学第三附属医院(广州中医药大学第三临床医学院、广州中医药大学附属骨伤科医院、广州中医药大学骨伤科研究所) | 一种肌少-骨质疏松症大鼠模型的构建方法 |
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