牙龈卟啉单胞菌感染通过Wnt通路调节牙周膜干细胞的成骨分化

目的 观察牙龈卟啉单胞菌(P.gingivalis)感染通过Wnt通路调节牙周膜干细胞(PDLSCs)成骨分化的作用。 方法 培养原代PDLSCs,分为常规处理的对照组、P.gingivalis感染的P.gingivalis组和P.gingivalis感染并用Wnt3a处理的P.gingivalis+Wnt3a组,成骨诱导后茜素红染色并检测A405值,Western blot检测Wnt通路分子的蛋白表达量,碱性磷酸酶(ALP)试剂盒检测ALP活力,PCR检测成骨标志基因Runt相关转录因子2(Runx2)、骨钙素(OCN)的mRNA表达量。 结果 与对照组比较,P.gingivalis组Wnt3a、βcatenin、pGSK3β的蛋白表达水平(0.33±0.07)、(0.27±0.08)、(0.44±0.09)以及成骨诱导后A405值(0.55±0.08)、ALP活力(20.14±6.54)U/mL和Runx2、OCN的mRNA表达量(0.45±0.09)、(0.51±0.07)均明显减少;与P.gingivalis组比较,P.gingivalis+Wnt3a组成骨诱导后A405值(0.89±0.15)、ALP活力(29.44±5.26)U/mL及Runx2、OCN的mRNA表达量(0.89±0.17)、(0.81±0.18)均明显增加。 结论 P.gingivalis感染能够抑制PDLSCs的成骨分化,抑制Wnt通路是可能的分子机制。     

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.     

DC‐CIK cells derived from ovarian cancer patient menstrual blood activate the TNFR1‐ASK1‐AIP1 pathway to kill autologous ovarian cancer stem cells

Ovarian cancer stem cells (OCSCs) are highly carcinogenic and have very strong resistance to traditional chemotherapeutic drugs; therefore, they are an important factor in ovarian cancer metastasis and recurrence. It has been reported that dendritic cell (DC)‐cytokine‐induced killer (CIK) cells have significant killing effects on all cancer cells across many systems including the blood, digestive, respiratory, urinary and reproductive systems. However, whether DC‐CIK cells can selectively kill OCSCs is currently unclear. In this study, we collected ovarian cancer patient menstrual blood (OCPMB) samples to acquire mononuclear cells and isolated DC‐CIK cells in vitro. In addition, autologous CD44+/CD133+ OCSCs were isolated and used as target cells. The experimental results showed that when DC‐CIK cells and OCSCs were mixed and cultured in vitro at ratios of 5:1, 10:1 and 50:1, the DC‐CIK cells killed significant amounts of OCSCs, inhibited their invasion in vitro and promoted their apoptosis. The qPCR and Western blot results showed that DC‐CIK cells stimulated high expression levels and phosphorylation of TNFR1, ASK1, AIP1 and JNK in OCSCs through the release of TNF‐α. After the endogenous TNFR1 gene was knocked out in OCSCs using the CRISPR/Cas9 technology, the killing function of DC‐CIK cells on target OCSCs was significantly attenuated. The results of the analyses of clinical samples suggested that the TNFR1 expression level was negatively correlated with ovarian cancer stage and prognosis. Therefore, we innovatively confirmed that DC‐CIK cells derived from OCPMB could secret TNF‐α to activate the expression of the TNFR1‐ASK1‐AIP1‐JNK pathway in OCSCs and kill autologous OCSCs.