摘要
骨髓基质干细胞移植在兔激素性股骨头坏死模型中成骨及成血管作用的评价
目的建立兔激素性股骨头坏死模型,体外培养和标记骨髓基质干细胞(MSCs),并通过血管内移植的方式将MSCs移植入股骨头供血动脉内,检测其在股骨头周围肌肉组织CD34及股骨头骨组织中的骨形态发生蛋白-2的表达,评价在激素性股骨头坏死模型中血管内移植骨髓基质干细胞(MSCs)的成骨及成血管作用。
方法
1、造模
日本大耳白兔30只,雄性12只,雌性18只,体重2.8±0.9kg。其中,前期实验10只,后期用作细胞移植的共20只。方法如下:从兔耳缘静脉内注射马血清2 mL,从第二天起于臀肌注射甲强龙(4 mg/kg),每周2次,共6周。前期实验死去3只兔子,存活7只。用作细胞移植的20只兔子,造模结束后死去5只,剩下15只随机分成两组,8只行右侧髂总动脉MSCs移植,作为移植组,其它7只不移植MSCs,作为对照组。
2、MSCs培养
无菌条件下,从20天龄的日本大耳白兔的髂骨和股骨中抽取骨髓,按1:1比例在骨髓液中加入PBS,反复抽吸、混匀,以1000 r/min速度离心10 min,去除上层的脂肪和杂质,再将骨髓液加入到密度为1.07g/L的、10mL的Percoll分离液中,以2500 r/min速度离心30 min,收集上层与Percoll分离液之间的单核细胞层。将抽提的骨髓单个核细胞加入到含低糖DMEM培养基的10%胎牛血清中培养,培养的孵箱条件为37℃、5%CO2,贴壁培养法分离、培养、传代。传代过程中,0.25%胰酶消化,吹管反复吹吸培养瓶瓶壁,直至细胞完全脱壁为止,PBS洗涤,按1:2传代。在移植前48小时,用BrdU(5mg/L)标记第三代MSCs。免疫组化检测MSCs的CD34、CD44表达,免疫组化及免疫荧光法检测细胞核的BrdU。移植前0.25%胰酶消化,吹管反复吹吸培养瓶瓶壁,PBS洗涤,离心,细胞计数,配制成3×10~6/mL浓度的悬液以备移植。
3、MSCs移植
分离移植组兔子的右侧股动脉,将导管尖端插入到髂总动脉水平,将BrdU标记的MSCs缓慢注入血管内,结扎右股动脉,继续饲养。
4、标本检测5周后取股骨头周围肌肉组织,部分标本制作冰冻切片,丙酮固定,加入一抗小鼠抗BrdU(1:300)和兔抗CD34(1:300),再分别加入小鼠IgG-Cy3 kit(1:100)和1:64 FITC标记的羊抗兔IgG(1:100),行BrdU和CD34免疫荧光双标记检测。另外标本10%甲醛固定,脱水、包埋、切片,厚5μm,加入一抗小鼠抗BrdU(1:100)和兔抗CD34(1:100),再分别加入相应二抗,行BrdU和CD34免疫组化双标记检测。取股骨头标本,10%甲醛固定,EDTA脱钙,脱水、固定、包埋、切片,厚5μm,加入一抗兔抗BMP-2(1:100)和小鼠抗BrdU(1:100),再分别加入相应二抗,行免疫组化BMP-2、BrdU双标记检测,并行HE染色以观察骨质变化。
结果
1、7例造模成活的日本大耳白兔股骨头均无明显塌陷,但6例出现弥漫性空骨陷窝或骨陷窝中细胞核萎缩,只有1例兔子空骨陷窝不到30%,但全部兔子均出现了骨髓腔内脂肪细胞肥大,哈弗管中脂肪细胞肥大聚集等变化,但未观察到微血管内血栓或脂肪栓子形成。造模成功率86%。
2、从幼兔骨髓中抽提的单个核细胞在培养瓶中贴壁生长,形状呈长梭形,免疫组化CD34阴性,CD44阳性,说明所培养的细胞不是骨髓造血干细胞,而是MSCs。
3、经过BrdU标记的细胞,体外免疫组化细胞核棕色,免疫荧光发红光,说明标记成功。
4、移植组股骨头周围肌肉组织BrdU、CD34双染色显示,该处肌肉组织中有大量BrdU阳性细胞,其中一部分胞浆CD34阳性,表明移植的一部分MSCs在股骨头颈部周围肌肉组织中向血管内皮细胞方向转化。冰冻切片BrdU-Cy3、CD34-FITC免疫双荧光也显示同样的结果。
5、MSCs移植组骨陷窝中可见BrdU阳性细胞,其胞浆中有BMP-2的表达,而对照组见大量空骨陷窝,BMP-2较少表达或不表达。
6、HE染色也显示,对照组骨陷窝内细胞核明显萎缩,接近空骨陷窝,哈弗氏管内细胞大量减少,而移植组的骨陷窝内细胞核较对照组组明显增大,哈弗氏管内细胞明显增加,但仍低于未造模未移植MSCs的正常兔子;未造模未移植MSCs的正常兔子的骨陷窝内细胞数量多,细胞核饱满,哈弗氏管内细胞多,核深染,活力正常。
结论
1、对于激素性ANFH,股骨头供血动脉内移植MSCs是可行的;
2、在股骨头供血血管内移植的MSCs,在局部肌肉组织中可向血管内皮细胞方向转化,在骨组织中可向成骨细胞方向转化,血管内移植骨髓基质干细胞有助于骨坏死的修复。
Objective
To establish the rabbit model of Avascular Necrosis of Femoral Head(ANFH). Mesenchymal stem cells(MSCs)were cultured and labeled in vitro,and then transplanted into the blood-supplying arteries around the femoral head.The expression of CD34 was checked up in the muscles around the femoral head and that of BMP-2 was also checked up in the bone tissues.To evaluate the effects of osteogenesis and angiogenesis after transplanting MSCs in the rabbit model of ANFH induced by corticosteroid.
Methods
(1)Model-making:Twenty Japanese white rabbits including twelve male ones and eighteen female ones were brought into the study,whose genders weren't limited and mean weight was 2.8±0.9 kg.Ten rabbits were used for preliminary study and twenty ones for formal experiment.Two milliliters of horse serum was injected from auricular veins,and then methylprednisolone(4 mg/kg)was injected from gluteous medius muscles,twice a week.It lasted for six weeks.Three rabbits were dead and seven ones were alive in the preliminary study.Of twenty rabbits used for MSCs transplanting,five rabbits were dead during model-making.Fifteen rabbits left were randomly divided into two groups,of which eight ones were used to transplanting MSCs from right iliac artery(transplanting group),while other ones weren't transplanted MSCs(control group).(2)MSCs culturing:Marrow was obtained from the ilium and the thighbone of the Janpanese rabbit(age of 20 days)under aseptic conditions.PBS was added into the marrow at the propotion of 1:1 and evenly mixed.The mixed marrow was centrifuged at the speed of 1000 rpm for ten minutes.The lipid and impurity in the upper layer were threw away.The marrow left was added into 10 milliliters of Percoll liquid(1.077 g/L) and then centrifuged at the speed of 2500 rpm.Monolayer was extracted from the layer between upper layer and Percoll liquid.These marrow-derived cells were cultured in L-DMEM supplemented wth 10%fetal bovine serum(FBS).The hatching room maintained at 37℃in 5%CO2.Afler primary culture,adhesive cells regenerate when they reach confluency.Meanwhile,cells were also cultured in a 6-well plates in complete medium.Cells were digested by 0.25%trypsin in the culture bottle and blown by a blowtorch.Then they were regenerated by a proportion of 1:2.48 hours before transplanting,BrdU(5 mg/L)was used to label the third generation of MSCs.CD34 and CD44 were respectively detected by immunohistochemistry for the 6-well cells.BrdU in cellular nucleus was also detected by immunofluorescence.Before thransplantation,cells labeled by BrdU were digested by 0.25%trypsin and blown by a blowtorch until cells were all away from the plastic culture bottle.Then they were rinsed by PBS and centrifuged.They were counted and suspensory fluid of 3×10~6 were prepared for transplantation.(3)Transplantation of MSCs:Right femoral artery was isolated in the transplanting rabbits and intubated.The catheter tip was up to the iliac artery level.MSCs labeled with BrdU were slowly injected into the arteries.The right femoral artery was ligated and the rabbit was sent back to the animal center.(4)Sample detection:5 weeks later,the musclular tissues around the right femoral head were obtained.A part of tissues were used for iecd section and fixed in acetone.A mouse anti-BrdU monoclonal primary antibody(1:300)and a rabbit anti-CD34 primary antibody(1:300)were added to the section,while a mouse IgG fluorescein-conjugated Cy3 kit secondary antibody(1:100)and a goat anti-rabbit IgG conjugated with FITC (1:100)were also added for a double immunofluorescent detection.Other tissues were fixed in 10%formaldehyde,then dehydrated in ethanol,embedded in paraffin,and sectioned at a thickness of 5μm.CD34 and BrdU were detected by immunohistochemistry similar to former immunofluorescent detection.Tissues of femoral head were also obtained,fixed in 10%formaldehyde,decalcified in EDTA,dehydrated in ethanol,embedded in paraffin,and also sectioned at a thickness of 5μm.A mouse anti-BrdU monoclonal primary antibody(1:100)and a rabbit anti-BMP-2 primary antibody(1:100)were added to the section,while respective secondary antibody was also added for a double immunohistochemistry detection.HE stain was used to detect osteogenic changes.
Results
(1)In the preliminary study,three rabbits were dead.Of seven rabbits left,no femoral head was found to cave in.Defuse empty lacunae or shrinked cellular nucleus were found in six rabbits,while only one rabbit was found that the number of empty lacunae was less than 30%.Lipocytes became enlarged in the bone marrow in all the rabbits.Lipocytes also became enlarged in Haverison canal.However,no thrombus or lipoembolus was found in the bone tissues.Well-made model was up to 86%.(2)MSCs extracted from the bone marrow of baby rabbit adhered to the wall of culturing bottle.They grew spindly.The expression of CD44 is positive while CD34 is negative.It showed that what we transplanted were MSCs other than Haematopoietic stem cells(HSC).(3)The nucleus labeled with BrdU showed brown color by immunohistochemistry,meanwhile it detected red light by immunofluorescence.It showed a successful labeling.(4)There were a lot of BrdU-positive cells in muscles around femoral head in transplanting group,of which many MSCs expressed CD34. The iced section also showed similar results by immunofluorecence.(5)BrdU-positive cells were also found in the lacunae of femoral head,in which BMP-2 was expressed in cellular plasm.(6)HE stain showed that cellular nuclei were evidently shrinked and close to empty laculae in control group.Cells evidenly decreased in Haverison canal.Meanwhile,cellular nuclei were much larger in transplanting group than those in contol group.Cells evidently increased in Haverison canal,but they were still less than those in normal rabbits without modeling.
Conclusion
(1)In the model of ANFH induced by corticosteroid,it is feasible to transplant MSCs from vessels;2 Transplanted marrow-derived stromal cells could reside at muscles around femoral head and osteogenic tissues.MSCs could be transdifferentiated into CD34-positive cells and BMP-2-positive cells.MSCs transplantation could be helpful to the angiogenesis and osteogenesis of osteonecrosis.
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