[Abstract ] Objective To explore the effective method of induction of Schwann cell-like differentiation in bone mesenchymal stem cell (BMSC)of adult rat in vitro.Methods Primary culture of Schwann cell and isolated culture of BMSC were separately conducted.According to different induction methods,the cells were divided into chemical induction group and co-culture induction group.The growth of Schwann cell and BMSC was observed under light microscope.These two kinds of cells were identified by immunofluorescence staining [detecting Schwann cell marker proteins:glial fibrillary acidic protein (GFAP) antibody and S-100 antibody] and flow cytometry.The shape and growth of cells in two groups were dynamically observed by light microscope.The induced differentiation was evaluated with immunofluorescence staining at 3 rd day after co-culture induction in the co-culture induction group and at 4 h and 1 st day after chemical induction in the chemical induction group.Results In the chemical induction group,the BMSC appeared typical Schwann cell-like morphology.The positive expression of GFAP antibody appeared at 4 h after preliminary induction.Meanwhile,the positive expression rate of GFAP and S-100 antibody was (80.9 ± 3.5)% and (59.0 ±1.1 )% at 1 st day after induction.The induced BMSC began to die at 2nd day after chemical induction and most of the induced BMSC had died at 3 rd day after chemical induction.At 3 rd day after co-culture induction,few induced BMSC showed obvious morphological changes like those in chemical induction group.The positive expression rate of GFAP and S-100 antibody was (89.8 ±2.4)% and (80.9 ±1.7)%. The positive expression rate of GFAP and S-100 antibody in the co-culture induction group was higher than those in the chemical induction group and the difference had statistical significance (all in P <0.01).Conclusions The co-culture induction not only has obvious effect on Schwann cell-like differentiation in BMSC,but also promotes the survival and proliferation of BMSC.Thus,co-culture induction is more safe and effective than chemical induction.

Objective To discuss the effect of bone marrow mesenchymal stem cell (BMSC)as the seed cell transplantation of tissue-engineered artificial nerve in the treatment of peripheral nerve injury. Methods BMSC was obtained from the bone marrow of adult rat through isolation and culture and combined with acellular nerve scaffold to construct ‘tissue-engineered artificial nerve’.After transplantation,rats were divided into two groups,the BMSC +acellular nerve conduit group(BMSC treatment group)and the empty cell conduit group(negative control group)with 5 rats in each group.Sciatic functional index (SFI)of the affected side of rats was compared between two groups at 2 weeks,4 weeks and 8 weeks after the surgery.Moreover,the sciatic conduction,recovery rate of tricipital muscle wet weight and other repair effects of the affected side were compared between two groups at 8 weeks after the surgery.Results The indicators of BMSC treatment group, including SFI assessed at 2 weeks,4 weeks and 8 weeks after the surgery as well as the sciatic conduction and recovery rate of tricipital muscle wet weight assessed at 8 weeks after the surgery,were better than those of the negative control group(all in P <0.05).Conclusions BMSC combined with tissue-engineered artificial nerve of acellular nerve scaffold can effectively promote nerve regeneration and function recovery.

Objective To investigate clinical efficacy of tissue-engineered artificial nerve grafts constructed by acellular nerve grafts combined with adult rat Schwann cell (SC)in the treatment of peripheral nerve injury.Methods SCs were isolated and cultured from the distal nerves of adult Sprague Dawley (SD) rats with 1-week Wallerian degeneration and then combined with acellular nerve grafts to construct tissue-engineered artificial nerve.All rats were divided into acellular nerve graft containing SCs (SC group)and nerve graft containing no cells groups (control group),five animals in each group.At 2-,4-and 8-week after surgery,sciatic function index (SFI)of the affected side was compared between two groups.At postoperative 8 weeks,nerve conduction of sciatic nerve of the injured side,recovery rate of triceps surae wet weight and other relevant parameters were equally compared between two groups.Results In the SC group,SFI of the affected side at 2-,4-and 8-week after surgery,nerve conduction of sciatic nerve at the injured side and recovery rate of triceps surae wet weight at postoperative 8 weeks were significantly better compared with those in the control group (all in P <0.05).Conclusions Combined use of adult rat SCs and acellular nerve grafts effectively repairs peripheral nerve defects and accelerates functional recovery of injured nerves.

Objective To establish a quality traceability evaluation method for the whole honeysuckle oral solution process by identifying and screening its anti-inflammatory quality markers.Methods UPLC/-TOF-MS was used to analyze the iridoids and phenolic acids in oral solution,and the correlations were constructed by molecular network technology.The HPLC fingerprints of multiple batches of oral solution were established,and similarity analyses were performed to identify key pharmacodynamic molecules.The key anti-inflammatory quality markers were confirmed by the NF-κB dual luciferase assay system.Further,the quantification of 12 quality markers of iridoids and phenolic acids in oral solution was established separately based on the dual-wavelength HPLC technique.The quality of the oral solution was evaluated by examining the extraction and transfer rate of quality markers during the processing of raw materials and preparations and thermal stability.Results A total of 9 iridoids and 6 phenolic acids were identified in the oral solution,and the possible conversion relationships between their components were depicted.Fingerprint analysis of 11 batches of oral liquids showed that the composition of their main peaks was the same,with a similarity of more than 90%.Among them,6 iridoids(loganic acid,secologanoside,secologanic acid,sweroside,secoxyloganin,secologanin)and 6 phenolic acids(neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,isochlorogenic B,isochlorogenic A,isochlorogenic C)exhibited NF-κB inhibitory activity,which were the main pharmacological components and could be used as quality markers.The traceability of the above 12 quality markers was investigated in a multi-batch process based on the dual-wavelength HPLC method.The thermal stability studies of the raw materials revealed that the contents of their total iridoids and phenolic acids remained stable.Still,some of them would be transformed between components.The production process of the oral solution was stable,and the transfer rates of the iridoids and phenolic acids during the extraction,concentration and preparation were over 76%and 63%,respectively.Conclusion The method is stable,reliable,easy to operate and can evaluate the full honeysuckle oral solution process,which provides an effective means for the quality control of honeysuckle herbs and preparations.

Background::Although the treatment of peripheral T-cell lymphoma (PTCL) has undergone advancements during the past several years, the response rate and long-term effects with respect to patients with PTCL remain unsatisfactory—particularly for relapsed or refractory (R/R) patients. This phase II trial was designed to explore the efficacy and safety of an all-oral regimen of chidamide plus prednisone, cyclophosphamide, and thalidomide (CPCT) for R/R PTCL patients who could not tolerate the standard chemotherapy for a variety of reasons.Methods::We conducted a multicenter phase II clinical trial in which we combined chidamide (30 mg twice weekly) with prednisone (20 mg daily after breakfast), cyclophosphamide (50 mg daily after lunch), and thalidomide (100 mg daily at bedtime) (the CPCT regimen) for a total of fewer than 12 cycles as an induction-combined treatment period, and then applied chidamide as single-drug maintenance. Forty-five patients were ultimately enrolled from August 2016 to April 2021 with respect to Chinese patients at nine centers. Our primary objective was to assess the overall response rate (ORR) after the treatment with CPCT.Results::Of the 45 enrolled patients, the optimal ORR and complete response (CR)/CR unconfirmed (CRu) were 71.1% (32/45) and 28.9% (13/45), respectively, and after a median follow-up period of 56 months, the median progression-free survival (PFS) and overall survival (OS) were 8.5 months and 17.2 months, respectively. The five-year PFS and OS rates were 21.2% (95% confidence interval [CI], 7.9-34.5%) and 43.8% (95% CI, 28.3-59.3%), respectively. The most common adverse event was neutropenia (20/45, 44.4%), but we observed no treatment-related death.Conclusion::The all-oral CPCT regimen was an effective and safe regimen for R/R PTCL patients who could not tolerate standard chemotherapy for various reasons.Trial Registration::ClinicalTrials.gov, NCT02879526.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.
Humans ; Transcriptional Activation ; RNA, Guide, CRISPR-Cas Systems ; Gene Expression Regulation ; CRISPR-Cas Systems/genetics*