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中华细胞与干细胞杂志(电子版) ›› 2023, Vol. 13 ›› Issue (06) : 331 -338. doi: 10.3877/cma.j.issn.2095-1221.2023.06.002

论著

组织获取位置和样本破损程度对胎盘来源造血干细胞质量的影响
崔光晶1, 王肇光1, 张雪梅2, 夏洁芳3, 李家梁1, 盛凯1, 任光强1, 刘东1,()   
  1. 1. 250000 济南,银丰生物工程集团有限公司
    2. 450000 郑州,河南银丰人类遗传资源库
    3. 7100003 西安,陕西省人类细胞资源库
  • 收稿日期:2024-01-18 出版日期:2023-12-01
  • 通信作者: 刘东
  • 基金资助:
    两链融合重点专项-陕西省人类细胞资源库及陕西区域细胞制备中心(2022QCY-LL-55)

Influence of tissue acquisition location and sample breakage on the quality of placental-derived hematopoietic stem cells

Guangjing Cui1, Zhaoguang Wang1, Xuemei Zhang2, Jiefang Xia3, Jialiang Li1, Kai Sheng1, Guangqiang Ren1, Dong Liu1,()   

  1. 1. YinFeng Biological Group, Jinan 250000, China
    2. Henan Yinfeng Human Resource Database, Zhengzhou 450000, China
    3. Shanxi Province Human Cell Resource Bank, Xi'an 7100003, China
  • Received:2024-01-18 Published:2023-12-01
  • Corresponding author: Dong Liu
引用本文:

崔光晶, 王肇光, 张雪梅, 夏洁芳, 李家梁, 盛凯, 任光强, 刘东. 组织获取位置和样本破损程度对胎盘来源造血干细胞质量的影响[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(06): 331-338.

Guangjing Cui, Zhaoguang Wang, Xuemei Zhang, Jiefang Xia, Jialiang Li, Kai Sheng, Guangqiang Ren, Dong Liu. Influence of tissue acquisition location and sample breakage on the quality of placental-derived hematopoietic stem cells[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2023, 13(06): 331-338.

目的

研究胎盘造血干细胞取样位置、破损程度与制备质量之间的关系,为临床胎盘样本收集及建立高质量的胎盘造血干细胞样本库提供指导和依据。

方法

根据胎盘造血干细胞制备位点取样后进行STR检测,分析不同取样位点的母血嵌合情况,确定精确取样位置。同时通过对胎盘破损程度进行分级,对不同破损级别的样本进行制备后的总有核细胞数量(TNC)、CD34+CD45dim流式、粒细胞-巨噬细胞集落形成单位(CFU-GM)等各项质量检测,分析不同破损程度的胎盘样本各项质量指标情况。根据是否满足正态分布,符合正态分布的多组比较采用One-way ANOVA分析法,两组间比较采用t检验,不符合正态分布的,两组间比较采用非参数检验。

结果

STR检测结果分析,与取样点1比较,取样点2外周血细胞嵌合率[(39.57 ± 4.56)%比(22.37 ± 3.61)%]升高,取样点3外周血细胞嵌合率[(2.17 ± 0.17)%比(22.37 ± 3.61)%]降低,差异有统计学意义(P < 0.01);与取样点3比较,取样点1和2外周血细胞嵌合率升高。与未破损组比较,三级破损组平均TNC数量[(5.52 ± 0.351)×108比(6.92 ± 0.83)×108]降低,差异具有统计学意义(P < 0.01)。与未破损组比较,一级破损组CD34+CD45dim细胞流式指标TNC的比例升高,二级、三级破损组占比降低,差异无统计学意义。与未破损组比较,二级、三级破损组CFU-GM集落平均总数降低,差异有统计学意义。

结论

不同的制备取样位置其母血嵌合率差异较大,尽量在靠近胎儿面位置取样有助于获得高质量的胎盘造血干细胞。同时通过对胎盘样本进行破损定级,胎盘破损程度越高,制备获取的胎盘TNC、CD34+CD45dim流式细胞数量、CFU-GM集落数量越低,其有效的造血干细胞数量降低,提示胎盘采集应尽量完整。

Objective

In order to analyze and study the relationship between the sampling location, degree of damage, and preparation quality of placental hematopoietic stem cells, it is necessary to optimize the precise sampling location of placental samples, establish criteria for examination grading, and provide guidance and a foundation for clinical collection of placental samples and establishment of a high-quality placental hematopoietic stem cell sample bank.

Method

After sampling the placental hematopoietic stem cell preparation site, STR detection was performed to analyze the mosaicism of maternal blood at different sampling sites and determine the precise location of sampling. Simultaneously, quality tests including grading the degree of placental damage, total nucleated cell number (TNC), CD34+CD45dim flow rate, and granulocyte- macrophage colony-forming unit (CFU-GM) were conducted on samples with varying levels of damage to analyze the quality indicators of placenta samples with different degrees of damage. Independent sample t-tests or non-parametric tests were utilized for data comparison and analysis.

Result

Analysis of STR test results, peripheral blood cell chimerism [ (39.57 ± 4.56) % vs (22.37 ± 3.61) %] was elevated at sampling site 2 compared to sampling site 1, while the peripheral blood cell chimerism rate at sampling point 3 [ (22.37 ± 3.61) % versus (2.17 ± 0.17) %] decreased. The difference was statistically significant (P < 0.01). Peripheral blood cell chimerism was elevated at sampling sites 1 and 2 compared with sampling site 3. Compared with the unbroken group, the mean number of TNCs was lower in the tertiary broken group [ (5.52 ± 0.35) × 108 compared with (6.92 ± 0.83) × 108], and the difference was statistically significant (P < 0.01). Compared with the unbroken group, the proportion of CD34+CD45dim cells to TNCs was elevated in the primary broken group and decreased in the secondary and tertiary broken group, with no statistically significant difference.Compared with the undamaged group, the average total number of CFU-GM in the second and third level damaged groups decreased, and the difference was statistically significant.

Conclusion

The mosaicism rate of maternal blood varies significantly across different sampling sites, highlighting the importance of sampling as close to the fetal surface as possible in order to obtain high-quality placental hematopoietic stem cells. Additionally, the grading of placenta samples revealed a negative correlation between the degree of damage and the number of TNC, CD34+CD45dim flow cells, CFU- GM colonies, and effective hematopoietic stem cells. This suggests that complete collection of placental tissue is crucial.

图1 胎盘取样位置示意及实际操作注:a图为胎儿胎盘示意图[8];b图为取样点3取样操作(靠近胎儿面);c图为取样点1取样操作(中间层位置);d图为取样点2取样操作(靠近母亲面);e图为组织剪碎操作;f图为酶消化后情况
图2 不同组织获取位置胎盘源细胞与母血嵌合比较注:与取样点1比较,aP < 0.05;与取样点2比较,bP < 0.05
表1 不同取样位置STR位点检测结果
图3 不同破损级别的样本制备后TNC数量比较注:与未破损组比较,aP < 0.05,ns为差异无统计学意义
图4 不同破损级别的样本制备后CD34+CD45dim流式比例比较
图5 不同破损级别样本制备后CD34+CD45dim细胞数量比较注:与未破损组比较,aP < 0.05ns为差异无统计学意义
图6 未破损组胎盘样本CD34+CD45dim细胞流式检测图片
图7 不同破损级别样本制备后CFU-GM集落总数比较注:aP < 0.01,bP < 0.001
图8 不同破损级别的样本制备后CFU-GM集落生长图注:a图为未破损组CFU-GM生长图;b图为一级破损组CFU-GM生长图;c图为二级破损组CFU-GM生长图;d图为三级破损组CFU-GM生长图
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