Shen Youliang, Zhang Jingjing, Yu Tengbo, Qi Chao
Department of Orthopaedics, Jiao Zhou Central Hospital of Qingdao City, Qingdao, 266300, China.
Orthopaedic Center, The Affiliated Hospital of Qingdao University, Qingdao, 266300, China.
Cytotechnology. 2018 Apr;70(2):783-791. doi: 10.1007/s10616-017-0183-3. Epub 2018 Jan 31.
The tumor suppressor PTEN is involved in the regulation of cell proliferation, lineage determination, motility, adhesion and apoptosis. Loss of PTEN in the bone mesenchymal stem cells (BMSCs) was shown to change their function in the repair tissue. So far, the CRISPR/Cas9 system has been proven extremely simple and flexible. Using this system to manipulate PTEN gene editing could produce the PTEN-Knocking-out (PTEN-KO) strain. We knocked out PTEN in MSCs and validated the expression by PCR and Western blot. To clarify the changes in proliferation, CCK-8 assay was applied. In support, living cell proportion was assessed by Trypan blue staining. For osteogenic and adipogenic induction, cells were cultured in different media for 2 weeks. Oil red staining and alizarin red staining were performed for assessment of osteogenic or adipogenic differentiation. The expression of Id4, Runx2, ALP and PPARγ was examined by qPCR and immunocytochemistry staining. The PTEN-KO strain was identified by sequencing. The PTEN-KO cells had an increased cell viability and higher survival compared with the wild type. However, decreased expression of Runx2 and PPARγ was found in the PTEN loss strain after induction, and consistently decreased osteogenic or adipogenic differentiation was observed by alizarin and oil red staining. Together, PTEN-KO strain showed an increased proliferation capability but decreased multi-directional differentiation potential. When BMSCs serve as seed cells for tissue engineering, the PTEN gene may be used as an indicator.
肿瘤抑制因子PTEN参与细胞增殖、谱系决定、运动、黏附和凋亡的调控。研究表明，骨间充质干细胞（BMSC）中PTEN的缺失会改变其在修复组织中的功能。到目前为止，CRISPR/Cas9系统已被证明极其简单且灵活。利用该系统进行PTEN基因编辑可产生PTEN基因敲除（PTEN-KO）菌株。我们在间充质干细胞中敲除PTEN，并通过PCR和蛋白质免疫印迹法验证其表达。为了阐明增殖变化，应用CCK-8检测法。此外，通过台盼蓝染色评估活细胞比例。为了进行成骨和成脂诱导，将细胞在不同培养基中培养2周。进行油红染色和茜素红染色以评估成骨或成脂分化。通过qPCR和免疫细胞化学染色检测Id4、Runx2、碱性磷酸酶（ALP）和过氧化物酶体增殖物激活受体γ（PPARγ）的表达。通过测序鉴定PTEN-KO菌株。与野生型相比，PTEN-KO细胞的细胞活力增加且存活率更高。然而，诱导后PTEN缺失菌株中Runx2和PPARγ的表达降低，并且通过茜素红和油红染色观察到成骨或成脂分化持续降低。总之，PTEN-KO菌株显示出增殖能力增加但多向分化潜能降低。当BMSC用作组织工程的种子细胞时，PTEN基因可作为一个指标。
