OBJECTIVE: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. METHODS: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. RESULTS: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. CONCLUSION Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Pregnancy ; Female ; Humans ; Rabbits ; Animals ; Vascular Endothelial Growth Factor A/metabolism* ; Fibronectins/metabolism* ; Collagen Type I/genetics* ; Tenascin/metabolism* ; Collagen/metabolism* ; Anterior Cruciate Ligament/surgery* ; Mesenchymal Stem Cells ; Tendons/metabolism* ; Fibroblasts/metabolism*

OBJECTIVE: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs. METHODS: The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot. RESULTS: The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-β1 single treatment group (P < 0.05). CONCLUSION A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.
Humans ; Transforming Growth Factor beta1/pharmacology* ; Hepatic Stellate Cells/pathology* ; Liver Cirrhosis/genetics* ; Collagen Type I/pharmacology* ; RNA, Messenger/metabolism*

OBJECTIVE: To observe the safety of donor NK cell infusions in the settings of hematopoietic stem cell transplantation and after consolidation chemotherapy in elderly patients with acute myeloid leukemia (AML). METHODS: Forty patients with AML were included, in which 21 patients aged over 60 years were at the stage of complete remission (CR) and 19 patients that received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mononucleated cells were isolated from peripheral blood from the donors (for allo-HSCT) or healthy immediate family members (elderly AML). The cells were seeded into the flasks pre-coated with NK cell specific activators, and expanded in media containing recombinant human IL-15 and IL-2 for 14 days. The cells were transfused intravenously after the identification of quality control. Trypan blue exclusion test was used for the determination of cell viability and counting. Flow cytometry analysis was performed to assess the surface antigenic profile. Seventy-eight infusions of the cell products were received by the elderly patients with AML after consolidation chemotherapy, 11 infusions were received by the patients during allo-HSCT and 32 infusions 3 moths after transplantation. The safety of cell therapy, body temperature, blood pressure and other indexes were observe during and 48 hours after cell transfusion. Meanwhile, the occurrence and severity of acute graft-versus-host disease (GVHD) were documented. RESULTS: Flow cytometry analysis showed that the proportion of NK cells (CD3^-CD56⁺) in the mononucleated cells before culture was (14.10±4.22)% (n=121), and the proportion increased dramatically up to (87.29±8.75)% (n=121) after culture for 14 days, the number of NK cells increased to 753.47±140.13 times (n=121). The doses of the infused NK cells was (7.58±2.50)×10⁷/kg per infusion. Moderate fever occurred in three cases after multiple infusions, and the temperature restored to normal on the same day after treatment. Fever was observed in one patient after every infusion of four times in total. The temperature reached to 38.5-39.0 ℃ and returned to normal within 1-2 hours after adequate antipyretic treatment, and then there was no discomfort. No GVHD was observed in the elderly AML patients, while 6 cases that received allo-HSCT developed moderate acute GVHD, among them grade I in 5 cases and grade II in 1 case. No other severe toxicities were observed. CONCLUSION NK cell products with a high-purity could be obtained by ex vivo expansion with this protocol. The transfusion of these expanded cells is generally safe in the elderly patients with AML that have received chemotherapy or patients that received hematopoietic stem cell transplantation.
Aged ; Graft vs Host Disease/prevention & control* ; Hematopoietic Stem Cell Transplantation ; Humans ; Killer Cells, Natural ; Leukemia, Myeloid, Acute/therapy* ; Middle Aged ; Recurrence

OBJECTIVE: To investigate the changes in bacterial flora in fecal samples, at the tumor loci and in adjacent mucosa in patients with colorectal cancer (CRC). METHODS: We collected fecal samples from 13 patients with CRC and 20 healthy individuals and tumor and adjacent mucosa samples from 6 CRC patients. The differences in bacterial composition between the fecal and mucosa samples were analyzed with 16S rDNA sequencing and bioinformatics methods. We also detected the total number of bacteria in the feces using flow cytometry, isolated and identified the microorganisms in the fecal and mucosa samples using common bacterial culture media. We further tested the effects of 7 isolated bacterial strains on apoptosis of 3 CRC cell lines using lactate dehydrogenase detection kit. RESULTS: The bacterial α-diversity in the feces of healthy individuals and in adjacent mucosa of CRC patients was significantly higher than that in the feces and tumor mucosa in CRC patients (P < 0.05). Lactobacillaceae is a specific bacteria in the feces, while Escherichia, Enterococcus, and Fusobacterium are specific bacteria in tumor mucosa of CRC patients as compared with healthy individuals. Cell experiment with3 CRC cell lines showed that Bacteroides fragilis isolated from the tumor mucosa of CRC patients produced significant inhibitory effects on cell proliferation (P < 0.0001), while the isolated strain Fusobacterium nucleatum obviously promoted the proliferation of the cell lines (P < 0.001). CONCLUSION The bacterial flora in the feces, tumor mucosa and adjacent mucosa of CRC patients is significantly different from that in the feces of healthy individuals, and the fecal flora of CRC patients can not represent the specific flora of the tumor mucosa. Inhibition of F. nucleatum colonization in the tumor mucosa and promoting B. fragilis colonization may prove beneficial for CRC treatment.
Bacteria ; Colorectal Neoplasms/pathology* ; Feces/microbiology* ; Gastrointestinal Microbiome ; Humans ; Intestinal Mucosa

Aim To investigate the effect of TRPC5 gene on the inflammation of cliabetie cardiomyopathy.Methods The biological functions of TRPC5 and the correlation between TRPC5 gene and other genes were analyzed by bioinformatics.Studies were performed in TRPC5 knockout ( TRPC5 ) and C57 mice.Mice were randomly divided into blank control and T2DM model groups, and the model was established by intraperitoneal injection of STZ (n = 10).The myocardial injury was detected by HE and Masson staining.Hie level of serum IL-1(3, IL-2, IL-6, IFN-7 and creatine kinase was examined by ELISA.Gene and protein expressions of IL-1(3, IL-2, IL-6 and TRPC5 were analysed by RT-PCR and Western blot respectively.Results By constructing the PPI network and analyses.the TRPC5 gene was identified to internet with a variety inflammatory genes and involved in immunity.The result of pathologieal section showed less myocardial damage and infiltrated immune cells in TRPC5 mice than in C57BL/6J mice.RT-PCR and serum results showed a lower expression of inflammatory factors in myocardium and serum obtained from TRPC5 model mice than in those obtained from C57BL/6J model mice.Conclusions TRPC5 participates in the development of dilated cardiomyopathy by regulating cardio- myocyte inflammation.

This study was designed to explore the protective effect and underlying mechanism of catalpol on hepatocyte apoptosis in nonalcoholic fatty liver disease (NAFLD). High fat diet (HFD) was used to establish NAFLD model in the in vivo experiment, and the procedures of the experiments and animal care protocol were approved by the Animal Care and Use Committee of Jianghan University. Human liver cancer cell line HepG2 was treated with palmitate (PA) to establish a lipid toxicity model in the in vitro experiments. The results showed that catalpol significantly decreased the contents of serum total glyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate transaminase (AST) in HFD-fed mice. Results of TUNEL staining and flow cytometry analyses revealed that catalpol significantly inhibited hepatocytes apoptosis in HFD-fed mice and PA-treated HepG2 cells. Moreover, catalpol treatment significantly reduced the endoplasmic reticulum stress-related protein expression levels of binding immunoglobulin protein (BiP), phosphorylated PKR-like endoplasmic reticulum kinase (p-PERK), inositol-requiring kinase 1α (IRE1α), and transcriptional factor activating transcription factor 6 (ATF6), and apoptosis-related protein expression levels of C/EBP homology protein (CHOP), phosphorylated c-Jun N-terminal kinase (p-JNK), and cleaved cysteinyl aspartate specific proteinases (caspases)-12, -9, and -3 in HFD-fed mice and PA-treated HepG2 cells. Furthermore, endoplasmic reticulum stress agonist tunicamycin (TM) significantly reversed the inhibitory effect of catalpol on protein expression levels of BiP, p-PERK, IRE1α, and ATF6, subsequently the inhibitory effect of catalpol on expression levels of CHOP, p-JNK, Bcl-2, Bax, and cleaved caspases (-12, -9, and -3) was also attenuated in PA-treated HepG2 cells. Taken together, these findings demonstrated that catalpol could inhibit hepatocytes apoptosis and had a significant protective effect on liver injury, and its mechanism might be related to the relief of endoplasmic reticulum stress.

The aim of this paper was to investigate the effect of Schizonepetae Herba and Saposhnikoviae Radix(wind medicine) on the expression of AQP4 and AQP8 in colonic mucosa in rats with ulcerative colitis(UC). A total of 35 healthy SD male rats were randomly divided into normal group(gavaged with normal saline), DSS model group, as well as low, middle, and high dose wind medicine groups(Schizonepeta and Saposhnikovia 1∶1, gavaged at dosages of 6, 12, and 24 g·kg~(-1)·d~(-1)), with 7 in each group. UC rat model was established by free drinking of 3% dextran sulphate sodium(DSS) solution for 10 days. At the end of the 10 th day after the treatment, mice were put to death to collect colonic mucosa. The length of colon was measured; the colonic mucosal injury index(CMDI) and pathological changes of colon were observed. ELISA method was used for measuring the content of serum IL-1, IL-8, and immunohistochemical method was used to measure AQP4, AQP8 protein expressions in colon mucosa. The expressions of AQP4, AQP8 mRNA were measured by Real-time PCR. As compared with the normal group, the length of colon tissue was significantly reduced(P<0.01), CMDI scores and pathological scores were significantly increased(P<0.01), the levels of serum IL-1 and IL-8 were significantly increased(P<0.05) in model group; the immunohistochemical results showed that the protein expressions of AQP4, AQP8 were lower; the color was light yellow or brown; AQP4, AQP8 mRNA expressions in colon mucosa were significantly decreased in model group(P<0.01). CMDI scores, pathological scores, and the levels of serum IL-1, IL-8 in high, middle, low dose wind medicine groups were obvious lower than those in the model group(P<0.01 or P<0.05); the protein expressions of AQP4, AQP8 were higher; the color was chocolate brown or dark brown; the length of colon tissue, and the expressions of AQP4, AQP8 mRNA were obvious higher in wind medicine groups(P<0.01 or P<0.05). Schizonepetae Herba and Saposhnikoviae Radix could significantly improve the symptoms and histopathology of UC model rats and accelerate the intestinal mucosal healing. The mechanism may be related with up-regulating the expression level of AQP4 and AQP8 in colonic mucosa.
Animals ; Apiaceae ; Aquaporin 4 ; Colitis, Ulcerative ; Colon ; Intestinal Mucosa ; Male ; Mice ; Plant Roots ; Rats

ObjectiveTo investigate the drug-resistant gene polymorphisms in Plasmodium falciparum imported from Equatorial Guinea to Shandong Province. MethodsFrom 2015 to 2016, blood samples were collected from imported P. falciparum malaria patients returning from Equatorial Guinea to Shandong Province, and genome DNA of the malaria parasite was extracted. The drug-resistant Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and K13 genes of P. falciparum were amplified using a PCR assay, followed by DNA sequencing, and the sequences were aligned. Results The target fragments of all 5 drug-resistant genes of P. falciparum were successfully amplified and sequenced. There were 72.8%, 18.6%, and 8.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfcrt gene, respectively, and all mutant haplotypes were CVIET (the underline indicates the mutation site). There were 20.0%, 61.4% and 18.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfmdr1 gene, respectively, and the mutant haplotypes mainly included YF and NF (the underlines indicate the mutation sites). There were 1.4%, 98.6%, and 0 of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfdhfr gene, respectively, and AIRNI was the predominant mutant haplotype (the underline indicates the mutation site). There were 1.4%, 94.3%, and 4.3% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfdhps gene, respectively, and SGKAA was the predominant mutant haplotype (the underline indicates the mutation site). The complete drug-resistant IRNGE genotype consisted of 8.6% of the Pfdhfr and Pfdhps genes, and the K13 gene A578S mutation occurred in 1.4% of the parasite samples. Conclusions There are mutations in the Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and K13 genes of P. falciparum imported from Equatorial Guinea to Shandong Province, with a low frequency in the Pfcrt gene mutation and a high frequency in the Pfmdr1, Pfdhfr, and Pfdhps gene mutations, and the K13 gene A578S mutation is detected in the parasite samples.

Objective To characterize the epidermal growth factor receptor (EGFR) gene in Schistosoma japonicum (SjEGFR gene) and investigate the role of the EGFR gene in regulating the growth, reproductive system, maturation and fecundity of S. japonicum. Methods Rapid amplification of cDNA ends (RACE) was performed to obtain the full length of the SjEGFR gene, and the SjEGFR gene expression was quantified in different developmental stages of S. japonicum using a quantitative real-time PCR (qPCR) assay. The tissue localization of the SjEGFR gene was detected in 22-day parasite using whole-mount in situ hybridization (WISH). Following RNA interference (RNAi)-induced knockdown of the SjEGFR gene, the worm length, pairing rate and worm burden of S. japonicum were measured, and the worm morphology was observed using optical microscopy and confocal microscopy. Results The SjEGFR gene was identified with a conserved tyrosine-kinase active site, and the SjEGFR gene expression was detected at various developmental stages in male and female parasites. WISH showed that the transcript of the SjEGFR gene was localized on the tegument and in the digestive organs of S. japonicum. RNAi-induced SjEGFR knockdown resulted in marked suppression of the worm growth, smaller size of male testicles that contained more immature spermatocytes, and apparent impairment of ovary and vitelline gland development. In addition, no eggs were found in the uterus of SjEGFR knocked-down female parasites, indicating the interruption of egg production. Conclusions Inhibition of SjEGFR expression may remarkably suppress the growth and maturation of S. japonicum, and interrupt the egg production.

CY-1-4 is a tryptanthrin derivative exhibiting antitumor activity. The solubility of CY-1-4 was poor and the corresponding mechanism needs further study. To solve this problem, we prepared nanoparticles encapsulated with CY-1-4 (CY-1-4 NPs) by nanoprecipitation method using poly(caprolactone) (PCL) and poly(ethylene glycol)-co-poly(ε-caprolactone) (PEG-PCL) as carriers to improve solubility. We then explored whether CY-1-4 NPs induced B16-F10 cytotoxicity via ferroptosis by determining the effect of CY-1-4 NPs on reactive oxygen (ROS) levels, repairing efficacy of lipid reactive oxygen inhibitor ferrostatin-1 and iron chelator deferoxamine (DFO), and potentiation of protoporphyrin (PPIX) induced B16-F10 cell death. The results showed that nanoparticlated strategy significantly improved solubility of CY-1-4. With the particle size about 116 nm, encapsulating efficacy was about 83% and the drug loading capacity was about 4.80%. Ferroptosis mechanistic studies indicated that CY-1-4 NPs could improve the ROS level in B16-F10 cells, whereas ferrostatin-1 and DFO could partly inhibited the cytotoxicity and PPIX could potentiated the cytotoxicity of CY-1-4 NPs in B16-F10 cells. These results showed that ferroptosis was one of the cell death mechanisms induced by tryptanthrin derivative CY-1-4 nanoparticle.