Spheroid-Like Cultures for Expanding Angiopoietin Receptor-1 (aka. Tie2) Positive Cells from the Human Intervertebral Disc

Lower back pain is a leading cause of disability worldwide. The recovery of nucleus pulposus (NP) progenitor cells (NPPCs) from the intervertebral disc (IVD) holds high promise for future cell therapy. NPPCs are positive for the angiopoietin-1 receptor (Tie2) and possess stemness capacity. However, the limited Tie2+ NPC yield has been a challenge for their use in cell-based therapy for regenerative medicine. In this study, we attempted to expand NPPCs from the whole NP cell population by spheroid-formation assay. Flow cytometry was used to quantify the percentage of NPPCs with Tie2-antibody in human primary NP cells (NPCs). Cell proliferation was assessed using the population doublings level (PDL) measurement. Synthesis and presence of extracellular matrix (ECM) from NPC spheroids were confirmed by quantitative Polymerase Chain Reaction (qPCR), immunostaining, and microscopy. Compared with monolayer, the spheroid-formation assay enriched the percentage of Tie2+ in NPCs’ population from ~10% to ~36%. Moreover, the spheroid-formation assay also inhibited the proliferation of the Tie2- NPCs with nearly no PDL. After one additional passage (P) using the spheroid-formation assay, NPC spheroids presented a Tie2+ percentage even further by ~10% in the NPC population. Our study concludes that the use of a spheroid culture system could be successfully applied to the culture and expansion of tissue-specific progenitors.     

Direct Reprogramming and Induction of Human Dermal Fibroblasts to Differentiate into iPS-Derived Nucleus Pulposus-like Cells in 3D Culture

Intervertebral disc (IVD) diseases are common spinal disorders that cause neck or back pain in the presence or absence of an underlying neurological disorder. IVD diseases develop on the basis of degeneration, and there are no established treatments for degeneration. IVD diseases may therefore represent a candidate for the application of regenerative medicine, potentially employing normal human dermal fibroblasts (NHDFs) induced to differentiate into nucleus pulposus (NP) cells. Here, we used a three-dimensional culture system to demonstrate that ectopic expression of MYC, KLF4, NOTO, SOX5, SOX6, and SOX9 in NHDFs generated NP-like cells, detected using Safranin-O staining. Quantitative PCR, microarray analysis, and fluorescence-activated cell sorting revealed that the induced NP cells exhibited a fully differentiated phenotype. These findings may significantly contribute to the development of effective strategies for treating IVD diseases.     

人碱性成纤维细胞生长因子在植物毛状根和毕赤酵母中的表达

目的在植物毛状根和毕赤酵母中表达人碱性成纤维细胞生长因子(Human basic fibroblast growth factor,hbFGF),并对发酵条件进行优化。方法将hbFGF基因分别克隆至表达载体pCAMBIA1301和pPICZα上,构建重组表达质粒pCAMBIA1301-hbFGF和pPICZα-hbFGF。通过发根农杆菌介导,将pCAMBIA1301-hbFGF质粒转入大豆毛状根;采用电转化法将质粒pPICZα-hbFGF转入毕赤酵母X-33菌株,PCR法筛选阳性转化子,诱导表达。筛选高效稳定表达株,发酵培养并优化发酵条件,SDS-PAGE和Western blot分析表达产物,并进行纯化。结果经酶切及测序证明两个重组表达质粒构建正确;经PCR鉴定证明hbFGF基因已整合入大豆毛状根及毕赤酵母基因组中;筛选出了在两种体系中发酵培养的最佳条件;hbFGF在大豆毛状根和毕赤酵母中的表达量分别占总蛋白的31%和42%,且均具有良好的反应原性;经纯化后,均可见相对分子质量约18000的单一条带。结论 hbFGF能够在大豆毛状根和毕赤酵母中高效表达,为其大规模工业化生产奠定了实验基础。     

川芎嗪对肝星状细胞增殖及结缔组织生长因子表达的影响

目的探讨川芎嗪(TMP)对肝星状细胞HSC-T6增殖及结缔组织生长因子(CTGF)表达的影响。方法不同剂量的TMP作用于HSC-T6细胞不同时间后,MTT法检测细胞的增殖;ELISA法检测Ⅰ型、Ⅲ型胶原和透明质酸(HA)的合成;West-ern blot法测定CTGF的表达。结果100～1000mg/L的TMP作用于HSC-T6细胞后,细胞的增殖明显受到抑制,且呈剂量依赖性;Ⅰ型、Ⅲ型胶原和HA的合成减少,CTGF的表达也减少,二者下降程度呈正相关(r分别为0.727、0.643和0.769)。结论TMP能够有效抑制肝纤维化形成,其可能的机制为抑制HSC增殖并降低CTGF的表达,从而阻断细胞内基质的形成。     

可溶性血管内皮生长因子受体2片段的表达及其对血管内皮细胞增殖的影响

目的探讨可溶性血管内皮生长因子受体2(sKDR)抑制血管内皮细胞增殖及在血管生成中的作用。方法提取脐静脉内皮细胞(HUVEC)总RNA,扩增KDR基因膜外1~4结构域,构建原核表达载体pQE40-KDR,转化E.coli M15,经IPTG诱导表达,镍离子柱亲和层析纯化后复性,用Western blot检测sKDR蛋白的表达,MTT比色法和鸡胚尿囊膜(CAM)试验分别检测其对HUVEC增殖的影响及其对血管生成的作用。结果经RT-PCR扩增得到了1150 bp左右的sKDR片段,并在pQE40原核表达系统中表达了sKDR蛋白,以包涵体形式存在。纯化后蛋白电泳呈现相对分子质量50000左右的单一条带,纯化蛋白占总蛋白的98%,蛋白含量为80μg/ml。Western blot证实其为重组sKDR蛋白。MTT检测结果显示,sKDR可抑制血管内皮生长因子(VEGF)刺激的HUVEC增殖,并阻滞VEGF诱导的CAM血管增生。结论已成功构建sKDR原核表达载体,并在大肠杆菌M15中获得表达,纯化的sKDR片段具有与VEGF结合的生物学功能,有望成为基因治疗肿瘤血管形成的理想靶点。     

DUSP-1 Induced by PGE2 and PGE1 Attenuates IL-1β-Activated MAPK Signaling,Leading to Suppression of NGF Expression in Human Intervertebral Disc Cells

The molecular mechanism of discogenic low back pain (LBP) involves nonphysiological nerve invasion into a degenerated intervertebral disc (IVD), induced by nerve growth factor (NGF). Selective cyclooxygenase (COX)-2 inhibitors are mainly used in the treatment of LBP, and act by suppressing the inflammatory mediator prostaglandin E₂ (PGE₂), which is induced by inflammatory stimuli, such as interleukin-1β (IL-1β). However, in our previous in vitro study using cultured human IVD cells, we demonstrated that the induction of NGF by IL-1β is augmented by a selective COX-2 inhibitor, and that PGE₂ and PGE₁ suppress NGF expression. Therefore, in this study, to elucidate the mechanism of NGF suppression by PGE₂ and PGE₁, we focused on mitogen-activated protein kinases (MAPKs) and its phosphatase, dual-specificity phosphatase (DUSP)-1. IL-1β-induced NGF expression was altered in human IVD cells by MAPK pathway inhibitors. PGE₂ and PGE₁ enhanced IL-1β-induced DUSP-1 expression, and suppressed the phosphorylation of MAPKs in human IVD cells. In DUSP-1 knockdown cells established using small interfering RNA, IL-1β-induced phosphorylation of MAPKs was enhanced and prolonged, and NGF expression was significantly enhanced. These results suggest that PGE₂ and PGE₁ suppress IL-1β-induced NGF expression by suppression of the MAPK signaling pathway, accompanied by increased DUSP-1 expression.