Chinese Journal of Tissue Engineering Research ›› 2022, Vol. 26 ›› Issue (24): 3814-3820.doi: 10.12307/2022.559
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Li Shidan1, Xing Wei2, Xie Xiaoyu1, Li Youbin1, Wang Shaochuan1, Fei Jun3
Received:
2021-07-21
Accepted:
2021-09-06
Online:
2022-08-28
Published:
2022-01-22
Contact:
Fei Jun, MD, Associate chief physician, Department of Emergency, Army Specialty Medical Center, Army Medical University, Chongqing 400002, China
About author:
Li Shidan, Master candidate, Attending physician, Department of Orthopedics, Army Specialty Medical Center, Army Medical University, Chongqing 400002, China
Supported by:
CLC Number:
Li Shidan, Xing Wei, Xie Xiaoyu, Li Youbin, Wang Shaochuan, Fei Jun. A new method for extracting human bone marrow mesenchymal stem cells and the comparison with traditional methods[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(24): 3814-3820.
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2.1 不同分离培养方法所提取细胞情况 2.1.1 不同离心方法骨髓分层情况 PBS组骨髓离心后分为4层,从下至上分别为:红细胞层、PBS及红细胞混合层、白膜层、血浆及脂肪层;而往PBS组中加入Ficoll分离液后,离心可分为5层,从下至上分别为:红细胞层、淋巴细胞分离液层、PBS及红细胞层、白膜层、血浆及脂肪层;外周血用PBS稀释混匀后加入Ficoll分离液中离心可分为4层:从下至上分别为:红细胞层、淋巴细胞分离液层、白膜层、PBS及血浆层,见图1A。从图中可见,对于骨髓标本,PBS浮于Ficoll分离液上层,而白膜层浮于PBS上层。若要获取骨髓中白膜层细胞,则直接吸取PBS上层细胞即可。而在外周血中,用PBS离心后白膜层介于PBS与Ficoll分离液之间,PBS与血浆均匀混合,不出现白膜层浮于PBS上层的情况。 2.1.2 原代细胞贴壁情况 将PBS组白膜层细胞接种至培养基后,培养基中可见中等量悬浮红细胞及少量脂肪泡。第3天镜下观察,有少量贴壁的梭形细胞,首次换液后红细胞显著减少,仍有少量脂肪泡残留。全骨髓组将骨髓接种至培养基后,培养基中可见大量悬浮红细胞及较多脂肪泡,换液前看不到贴壁细胞,第7天首次换液后仍可见少量红细胞及较多脂肪泡,可见部分贴壁梭形细胞,见图1B。首次换液前后,对可见的梭形细胞计数,PBS组细胞明显多于全骨髓组,见图1C。"
2.2 细胞形态、大小及细胞骨架 相差显微镜下可见两组细胞形态、大小较为均一;结晶紫染色后可见大量长梭形细胞,细胞较为细长,中间粗壮,两端纤细,触角较少;少部分细胞呈扁平状,触角稍多。鬼笔环肽及DAPI染色见两组细胞均为单核细胞,细胞骨架完整,胞核及蛋白纤维网架体系清晰可见,见图2A。PBS组所提取细胞长宽比为10.876±3.093,全骨髓组所提取细胞长宽比为9.485±3.349,两组相比差异无显著性意义(t=0.915 5,P=0.373 5);PBS组所提取细胞平均面积为(1 342.665±290.731) μm2,全骨髓组所提取细胞平均面积为(1 679.665±466.343) μm2,两组相比差异无显著性意义(t=1.840,P=0.084 4),见图2B。"
2.5 细胞集落形成及细胞生长曲线 2.5.1 集落形成 细胞接种于6孔板后24 h行结晶紫染色即可看到集落形成。相对于24 h,在48 h及72 h可见细胞集落增大,集落数量增多,见图5A。每一个6孔板空格中,24 h PBS组细胞集落数为(5.333±0.577)个,全骨髓组为(8.000±1.000)个,两组相比差异有显著性意义(t=4.000,P=0.016 1);48 h PBS组平均集落数为(14.000±1.000)个,全骨髓组为(17.000±1.000)个,两组相比差异有显著性意义(t=3.674,P=0.021 3);72 h PBS组平均集落数为(20.667±1.528)个,全骨髓组为(25.000±2.000)个,两组相比差异有显著性意义(t=2.982, P=0.040 6),见图5B。 2.5.2 细胞生长曲线 在72 h之前,两组细胞吸光度值低,生长曲线平缓上升,细胞呈缓慢生长状态。在72 h之后,细胞生长曲线斜率增大,细胞快速生长,于120 h细胞长满96孔板。全骨髓组细胞生长速度快于PBS组:24 h时PBS组细胞吸光度值为0.058±0.010,全骨髓组为0.061±0.004,两组相比差异无显著性意义(t=0.715 8,P=0.494 5);48 h时PBS组细胞吸光度值为0.113±0.010,全骨髓组为0.139±0.010,全骨髓组高于PBS组(t=4.079,P=0.003 5);72 h时PBS组细胞吸光度值为0.192±0.031,全骨髓组为0.247±0.020,全骨髓组高于PBS组(t=3.325,P=0.010 5);96 h时PBS组细胞吸光度值为0.549±0.041,全骨髓组为0.696±0.089,全骨髓组高于PBS组(t=3.353,P=0.010 0);120 h时PBS组细胞吸光度值为1.653±0.123,全骨髓组为1.748±0.127,两组相比差异无显著性意义(t=1.199,P=0.256 0),见图5C。"
2.6 细胞迁移能力比较 2.6.1 细胞划痕实验 随着时间推移,两组划痕区域周边细胞逐渐向划痕区域生长。24 h之前细胞迁移速率较快,划痕区域有80%左右细胞长入,24 h之后细胞长入速率减缓,见图6A。在12 h时,PBS组细胞愈合率为0.344±0.032,全骨髓组愈合率为0.525±0.015,全骨髓组愈合率大于PBS组(t=8.980,P=0.000 9);在24 h时,PBS组细胞愈合率为0.814±0.061,全骨髓组愈合率为0.836±0.061,两组相比差异无显著性意义(t=0.440 7,P=0.682 2);36 h时,PBS组细胞愈合率为0.863±0.055,全骨髓组愈合率为0.904±0.031,两组相比差异无显著性意义(t=1.157,P=0.311 7),见图6B。 2.6.2 Transwell实验 24 h时,两组均有少量细胞穿过Transwell 小格,从上室迁移至下室。随着时间变化,迁移细胞逐渐增多,72 h有大量细胞迁移至小室下层,见图6C。24 h时,PBS组平均每个100×视野,有(51.33±6.658)个迁移细胞,全骨髓组有(108.667±3.512)个迁移细胞,全骨髓组迁移细胞数多于PBS组(t=13.19,P=0.000 2);48 h时,PBS组迁移细胞数为(393.667±65.310)个,全骨髓组迁移细胞数为(594.667±35.388)个,全骨髓组迁移细胞数多于PBS组(t=4.687,P=0.009 4);72 h时,PBS组迁移细胞数为(1 025.333±110.961)个,全骨髓组迁移细胞数为 (1 145.333±152.753)个,两组相比差异无显著性意义(t=1.101,P=0.332 7),见图6D。"
2.7 成骨分化过程中成骨标志物表达 未分化的两组骨髓间充质干细胞基本不表达成骨相关标志物BMP2、IBSP、Runx2、COL1A1、ALP、BGLAP。成骨分化诱导分化过程中,随着时间推移,相应的成骨标志物表达增高。第4天时,全骨髓组表达量高于PBS组的基因有Runx2( t=13.89,P=0.000 2)、COL1A1( t=15.88,P=0.000 1)、ALP( t=4.250,P=0.013 2);PBS组高于全骨髓组的基因为BGLAP(t=5.791,P=0.004 4);第8天时,全骨髓组表达量高于PBS组的基因有BMP2( t=2.852,P=0.046 3)、IBSP( t=9.118,P=0.000 8)、COL1A1( t=6.710,P=0.002 6)。其余时间,两组相比基因表达量差异无显著性意义,见图7。"
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