Objective To develop new methods to cultivate, retrieve and purify mouse mesenchymal stem cells (mMSCs). Methods Bone marrow was collected from 2-month-old Kunming mice by flushing femurs and tibias with complete medium of DMEM-LG. Cells were plated in a Petri dish. After 24 hours, non-adherent cells were removed by two to three washes with PBS, adherent cells were further cultured in complete medium and retrieved by trypsinisation with 0.25% trypsin for 5 min at 37 ℃. The treated adherent cells were cultivated with 3?dilution for further generations. CD11b-negative cells were retrieved from the collected adherent cells of 3rd generation by using immunomagnetic microbeads, and continued to be cultured in complete medium. After the cultured cells were retrieved, their morphology and their ability of osteoblastic differentiation and adipocytic differentiation were examined. Results Most of mMSCs from 1st generation were of shuttle shape, some of irregular shape. After treatment with magnetic microbeads and several generations, mMSCs were of spindle, star and irregular shape. These cells were of rich cytoplasma, clear nucleolus, and grew in parallel or vortex. The cultured adherent cells from the first and subsequent generations had plenty of CD11b-positive blooding-making cells. After 20-day osteoblastic induction, mMSCs differentiated into bone cells, which showed orange phosphate in extracellular matrix by Alizarin red S staining. mMSCs could differentiate into lipocytes. The size of cells increased along with fat-developing induction period. These cells showed many orange fatty follicles with O Red Oil dyeing. Conclusion Pure mMSCs can not be retrieved by either adhering method or generation cultivation method separately. The combined methods of adhering, immunomagnetic microbeads, and serial subcultivation is effective in vitro in retrieve mMSCs.

ObjectiveTo investigate the association of BANK1 single nucleotide polymorphisms (SNPs) with rheumatoid arthritis(RA) in Chinese Han. MethodsTwo hundreds and twenty-one RA patients and 310 healthy controls who were Chinses Han population from Huashan Hopital and Changzheng Hospital in Shanghai,China were included.DNAs were extracted from peripheral whole blood for study.Samples were genotyped for three variants rs10516487,rs17266594 and rs3733197 in BANK1 by unlabelled probe high resolution melting (HRM) assay.The genotype frequencies of the detected polymorphisms were analyzed in relation to RA and the production of autoantibodies in RA patients.ResultsThe Tr genotype frequency was much higher in RA patients than in healthy controls(X2=6.241,P=0.044).The frequencies of rs10516487 G allele,rs17266594 T allele and rs3733197 G allele were increased among RA patients compared with healthy controls,although they didn't reach statistical significance.The rs10516487 and rs17266594 were found in strong linkage disequilibrium(D'=0.993,r2=0.985).And also the major TGG haplotype of 3-SNP was significantly associated with RA patients[P=0.037,OR =1.345,95％CI (1.018-1.776)].ConclusionBANK1 rs17266594 polymorphism is susceptible to RA,while rs10516487 and rs17266594 are linked in Chinese Han population.BANK1 SNPs TGG haplotype may contribute to RA susceptibility,too.

Objective To explore the influence of excessive PTEN expression to fibroblast cycle and collagen secretion induced by LPS.Methods Normal skin fibroblast in the patient with hyperplastic scar were cultured in vitro.When the primary culture was close to 80% fusion,the digestive passage was performed,cultured to the third generation.LPS(0.5 μg/mL) was adopted to stimulate the third generation of normal skin fibroblasts.Defective adenovirus carrying PTEN gene was transfected to the third passage fibroblasts after LPS stimulation.Flow cytometer was adopted to detect the cell cycle.ELISA method was adopted to detect the secreted collagen amont.Results Excessive PTEN expression could inhibit the increase of G2M cell cycle induced by LPS.LPS stimulation could increase the secretion of collagen in skin fibroblasts,yet excessive PTEN expression could inhibit the secretion of collagen induced by LPS.Conclusion LPS could increase the amont of fibroblasts on G2M cell cycle and secretion of collagen,yet excessive PTEN expression can inhibit the effect.

BACKGROUND:Percutaneous vertebroplasty and percutaneous kyphoplasty in the treatment of osteoporotic vertebral compression fracture have obtained good outcomes, because the traditional method is invalid, but there are a variety of choices in operation time, anesthesia, surgical approach and method, and each method has its advantages and disadvantages. OBJECTIVE:To investigate the effect and preponderance of the manual reduction combined with unilateral percutaneous vertebroplasty under general anesthesia in the treatment of osteoporotic vertebral compression fractures. METHODS:A total of 53 patients with single vertebral osteoporotic vertebral compression fractures, who were treated with percutaneous vertebroplasty, were retrospectively analyzed from July 2012 to December 2014. The new method group (32 cases) received manual reduction, underwent unilateral pedicle puncture and bone cement injection during unilateral percutaneous vertebroplasty under general anesthesia. The conventional method group (21 cases) received conventional percutaneous vertebroplasty. RESULTS AND CONCLUSION: There was an average of 6-month folow-up (3-14 months). Significant differences in visual analogue scale scores, vertebral compression ratio and kyphosis Cobb’s angle were detected in the new method and the conventional method groups at 3 days post surgery and during final folow-up compared with before surgery (P < 0.01). No significant difference in visual analogue scale scores was found between the two groups (P > 0.05). Compared with the conventional method group, postoperative vertebral compression ratio, kyphosis Cobb’s angle and bone cement leakage rate were significantly lower in the new method group (P < 0.01). Results verified that the new method combined with the advantages of percutaneous vertebroplasty and percutaneous kyphoplasty, the advantages of unilateral and bilateral puncture approach. The new method can correct kyphosis deformity, effectively recover the vertebral height and physiological curvature and the puncture is safe. Simultaneously, the leakage rate of bone cement is reduced, and the distribution of bone cement is ideal.

Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment.Oral squamous cell carcinoma(OSCC),a representative hypoxic tumor,has a heterogeneous internal metabolic environment.To clarify the relationship between different metabolic regions and the tumor immune microenvironment(TME)in OSCC,Single cell(SC)and spatial transcriptomics(ST)sequencing of OSCC tissues were performed.The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data.The metabolic activity of each spot was calculated using scMetabolism,and k-means clustering was used to classify all spots into hyper-,normal-,or hypometabolic regions.CD4T cell infiltration and TGF-β expression is higher in the hypermetabolic regions than in the others.Through CellPhoneDB and NicheNet cell-cell communication analysis,it was found that in the hypermetabolic region,fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts(iCAFs),and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12.The secretion of CXCL12 recruits regulatory T cells(Tregs),leading to Treg infiltration and increased TGF-β secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment.This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC,ST and TCGA bulk data,and highlights potential targets for therapy.

Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment.Oral squamous cell carcinoma(OSCC),a representative hypoxic tumor,has a heterogeneous internal metabolic environment.To clarify the relationship between different metabolic regions and the tumor immune microenvironment(TME)in OSCC,Single cell(SC)and spatial transcriptomics(ST)sequencing of OSCC tissues were performed.The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data.The metabolic activity of each spot was calculated using scMetabolism,and k-means clustering was used to classify all spots into hyper-,normal-,or hypometabolic regions.CD4T cell infiltration and TGF-β expression is higher in the hypermetabolic regions than in the others.Through CellPhoneDB and NicheNet cell-cell communication analysis,it was found that in the hypermetabolic region,fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts(iCAFs),and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12.The secretion of CXCL12 recruits regulatory T cells(Tregs),leading to Treg infiltration and increased TGF-β secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment.This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC,ST and TCGA bulk data,and highlights potential targets for therapy.

Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment.Oral squamous cell carcinoma(OSCC),a representative hypoxic tumor,has a heterogeneous internal metabolic environment.To clarify the relationship between different metabolic regions and the tumor immune microenvironment(TME)in OSCC,Single cell(SC)and spatial transcriptomics(ST)sequencing of OSCC tissues were performed.The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data.The metabolic activity of each spot was calculated using scMetabolism,and k-means clustering was used to classify all spots into hyper-,normal-,or hypometabolic regions.CD4T cell infiltration and TGF-β expression is higher in the hypermetabolic regions than in the others.Through CellPhoneDB and NicheNet cell-cell communication analysis,it was found that in the hypermetabolic region,fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts(iCAFs),and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12.The secretion of CXCL12 recruits regulatory T cells(Tregs),leading to Treg infiltration and increased TGF-β secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment.This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC,ST and TCGA bulk data,and highlights potential targets for therapy.

Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment.Oral squamous cell carcinoma(OSCC),a representative hypoxic tumor,has a heterogeneous internal metabolic environment.To clarify the relationship between different metabolic regions and the tumor immune microenvironment(TME)in OSCC,Single cell(SC)and spatial transcriptomics(ST)sequencing of OSCC tissues were performed.The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data.The metabolic activity of each spot was calculated using scMetabolism,and k-means clustering was used to classify all spots into hyper-,normal-,or hypometabolic regions.CD4T cell infiltration and TGF-β expression is higher in the hypermetabolic regions than in the others.Through CellPhoneDB and NicheNet cell-cell communication analysis,it was found that in the hypermetabolic region,fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts(iCAFs),and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12.The secretion of CXCL12 recruits regulatory T cells(Tregs),leading to Treg infiltration and increased TGF-β secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment.This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC,ST and TCGA bulk data,and highlights potential targets for therapy.

Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment.Oral squamous cell carcinoma(OSCC),a representative hypoxic tumor,has a heterogeneous internal metabolic environment.To clarify the relationship between different metabolic regions and the tumor immune microenvironment(TME)in OSCC,Single cell(SC)and spatial transcriptomics(ST)sequencing of OSCC tissues were performed.The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data.The metabolic activity of each spot was calculated using scMetabolism,and k-means clustering was used to classify all spots into hyper-,normal-,or hypometabolic regions.CD4T cell infiltration and TGF-β expression is higher in the hypermetabolic regions than in the others.Through CellPhoneDB and NicheNet cell-cell communication analysis,it was found that in the hypermetabolic region,fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts(iCAFs),and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12.The secretion of CXCL12 recruits regulatory T cells(Tregs),leading to Treg infiltration and increased TGF-β secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment.This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC,ST and TCGA bulk data,and highlights potential targets for therapy.

Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment.Oral squamous cell carcinoma(OSCC),a representative hypoxic tumor,has a heterogeneous internal metabolic environment.To clarify the relationship between different metabolic regions and the tumor immune microenvironment(TME)in OSCC,Single cell(SC)and spatial transcriptomics(ST)sequencing of OSCC tissues were performed.The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data.The metabolic activity of each spot was calculated using scMetabolism,and k-means clustering was used to classify all spots into hyper-,normal-,or hypometabolic regions.CD4T cell infiltration and TGF-β expression is higher in the hypermetabolic regions than in the others.Through CellPhoneDB and NicheNet cell-cell communication analysis,it was found that in the hypermetabolic region,fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts(iCAFs),and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12.The secretion of CXCL12 recruits regulatory T cells(Tregs),leading to Treg infiltration and increased TGF-β secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment.This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC,ST and TCGA bulk data,and highlights potential targets for therapy.