BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5％ CO2,37℃ thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse,and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter,whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P<0.0001).(3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P<0.0001).(4) qPCR and western blot assay revealed that the mRNA (P<0.0001) and protein expression levels (P<0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse.(5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.

Objective To establish primary breast cancer cell lines from patient tissues and offer a new cancer cell model for basic research.Methods Breast cancer biopsy tissues were digested with typeⅡcollagenase and cultured in BCMI medium.When the cells proliferated rapidly,the medium was switched to DMEM.STR genotyping was per-formed to identify specific genetic markers of the four primary breast cancer cell lines.Colony expansion assays and sphere formation assays were conducted to analyze its tumorigenicity.Real-time PCR and Western blot experiments were used to analyze the expression of the epithelial-mesenchymal transition(EMT)molecule markers.Migration and invasion assays were performed to assess the metastatic potential of the primary breast cancer cells.Results We es?tablished four primary breast cancer cell lines:BC25#,BC51#,BC56#,and BC57#.These cell lines were cultured in DMEM medium,passaged multiple times and tagged with details about their clinical past.STR genotyping identified specific genetic markers for each of the four primary breast cancer cell lines.Clonogenic and sphere formation assays revealed that the four lines have a stronger tumor?forming capability compared to the classic breast cancer cell line T?47D.Real?time PCR and Western blot experiments showed that,compared to T?47D,the four primary breast cancer cell lines have decreased E?cadherin expression and increased vimentin expression.Migration and invasion assays indicated that BC25#had a higher metastatic potential than the traditional breast cancer cell line T?47D.Conclusions Four primary breast cancer cell lines,BC25#,BC51#,BC56#and BC57#are successfully estab?lished,which may act as new cancer cell model for laboratory research of breast cancer.

BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5％ CO2,37℃ thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse,and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter,whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P<0.0001).(3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P<0.0001).(4) qPCR and western blot assay revealed that the mRNA (P<0.0001) and protein expression levels (P<0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse.(5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.

Objective To investigate the expression of long non-coding RNA(LncRNA)PICSAR in ovarian canc-er,and explore the effects of LncRNA PICSAR on the proliferation,migration,invasion and apoptosis of ovarian cancer cells as well as its possible mechanism of action.Methods The expression levels of LncRNA PICSAR in o-varian cancer tissue and cell line A2780,OVCAR-3,HO-8910 and normal ovarian tissue and cell line IOSE386 were detected by fluorescence quantitative PCR.Ovarian cancer cell lines with the highest expression of LncRNA PICSAR were divided into control group and experimental group,and transfected with negative control small inter-fering RNA(siRNA-NC)or PICSAR knockout small interfering RNA(siRNA-PICSAR)by liposome transfection technique,respectively.The effects of LncRNA PICSAR knockdown on the invasion,migration,proliferation and apoptosis of ovarian cancer cells were analyzed by cell counting assay(CCK-8),clonogenic assay,scratch assay,transwell assay and flow cytometry and so on.The expression levels of autophagy related proteins and apoptosis-re-lated proteins in each group were determined by Western blot.Ovarian cancer cells were treated with rapamycin and hydroxychloroquine as autophagy activator and inhibitor,and Western blot assay was used to detect apoptosis.Results The expression level of LncRNA PICSAR in ovarian cancer tissues was higher than that in normal ovarian tissues.Compared with IOSE386 cell line,LncRNA PICSAR expression levels in ovarian cancer cell lines HO-8910,OVCAR-3 and A2780 increased.Compared with the si-NC group,the proliferation,invasion and migration ability of ovarian cancer cells in si-PICSAR group decreased,and the apoptosis rate increased.The autophagy level of ovarian cancer cells in si-PICSAR group was lower than that in si-NC group.After transfection with siRNA-PIC-SAR,rapamycin activated autophagy to reduce apoptosis,while hydroxychloroquine inhibited autophagy to promote apoptosis.Conclusion LncRNA PICSAR is highly expressed in ovarian cancer tissues and cell lines,and the ma-lignant biological behavior of ovarian cancer cells can be inhibited by knockout of LncRNA PICSAR.The knock-down of LncRNA PICSAR may promote the apoptosis of ovarian cancer cells by regulating autophagy.

Background and purpose:In 2016 the National Cancer Institute(NCI)decided stopping to use NCI-60 cell lines for drug screening,suggesting that tumor cell lines were losing their value as a tool for drug discovery and basic research.The reason for NCI-60 cells'retirement'was that the preclinical studies based on traditional cellular and animal models did not obtain the corresponding expected efficacy in clinical trials.Since the major cancer behaviors,such as proliferation and metastasis,are fundamentally altered with long-term culture,the tumor cell lines are not representative of the characteristics of cancer in patients.Currently,scientists hope to create a new cancer model that are derived from fresh patient samples and tagged with details about their clinical past.Our purpose was to create patient-derived breast cancer primary cell lines as new cancer model for drug screening and basic research.Methods:Breast cancer tissues were collected in the Department of Breast Surgery,Affiliated Hospital of Guizhou Medical University.The collection of tumor tissue samples was approved by the Ethics Committee of the Affiliated Hospital of Guizhou Medical University(approval number:2022 ethics No.313),and the collection and use of tumor tissues complied with the Declaration of Helsinki.The primary breast cancer cell lines were isolated from the patient's breast cancer tissues and cultured in BCMI medium.After the cells proliferated,the media were replaced with DEME medium.Cell line STR genotyping was done to determine cell-specific genetic markers and identification.Clone formation assay and transplantation assay were done to analyze the ability of breast cancer primary cell lines to form tumors.Results:We created 6 primary breast cancer cell lines.The 6 primary breast cancer cell lines from the patients were tagged with the definitively clinicopathological features,clinical diagnosis,therapeutic regimens,clinical effectiveness and prognostic outcomes.The STR genotyping assays identified the genetic markers and determined the identities of the 6 primary breast cancer cell lines.Clone formation assays and transplantation assay showed that the proliferative capacities of the patient-derived primary breast cancer cell lines were significantly greater compared with the conventional breast cancer cell lines.Conclusion:We created a panel of 6 patient-derived primary breast cancer cell lines as new cancer model for drug screening and basic research in breast cancer.

Precancerous lesions of gastric cancer is a key stage in the development of gastric cancer.The reprogramming of glucose metabolism is a prominent feature of precancerous lesions of gastric cancer.Hypoxic microenvironment and hypoxia-inducible factors are important factors influencing the occurrence of glucose metabolic reprogramming.This article summarized the relationship between hypoxic microenvironment and the reprogramming of glucose metabolism in precancerous lesions of gastric cancer,and concluded the relevant research on TCM compounds and effective components to improve hypoxic microenvironment and further regulate glycolysis for the treatment of this disease.It was concluded that the mechanism may be the inhibition of angiogenesis,regulation of signaling pathways and key proteins of glycolysis,expression of multiple enzymes,reduction of lactate secretion,inhibition of cell malignant proliferation and invasion.It explored the mechanism of Chinese materia medica in improving hypoxic microenvironment and regulating glycolysis,so as to provide reference for the prevention and treatment of precancerous lesions of gastric cancer.

Objective:To evaluate the ability of light dispersive colorimetry to detect hemoglobin (Hb) in lipid blood samples and its feasibility as an alternative to plasmapheresis commonly used in the laboratory.Methods:Routine blood samples of 276 inpatients in Fujian Provincial Hospital from July 2020 to July 2021 were collected. Routine blood samples of 276 inpatients were collected. There were 169 males and 107 females, aged from 16 to 97 years. 183 non-lipid blood samples and 93 lipid blood samples were collected.(1) One case each of low, medium and high Hb value in non-lipid blood and lipid blood samples were collected, and the precision and the linearity of light scattering method was detected.(2)Non-lipid blood samples divided into Hb low-value group, median-value group and high-value group, which were measured by light scattering method and colorimetric method to compare Hb values. (3)Non-lipid blood samples were divided into Hb low-value group, median-value group and high-value group. Plasma exchange was carried out with different concentrations of fat emulsion. The bias and linearity of Hbc2 and Hbc1, Hbo2 and Hbc1 were analyzed by MedCalc19.1 software. The Hbc2 and Hbc1 bias ( CV%) and Hbo2 and Hbc1 bias ( CV%) were calculated. T test was used to analyze the influence of different concentrations of triglyceride on Hb bias.(4)Blood samples were divided into Hb low-value group, median-group and high-value group. The Hb of light scattering method was compared with the colorimetric method after plasma exchange. Results:(1)The intrabatch precision of light scattering method for non-lipid blood and lipid blood specimens was within the allowable range ( CV<1.5%), and the good linearity ( R2=1.000).(2)The bias of Hb measured by light scattering method and colorimetric method in the three groups was below 3.5%(-0.58±2.34,0.16±1.52,1.15±1.56), within the allowable total error range. The two methods have the equivalence and good linear relationship ( r=0.999).(3)The concentrations of Hbo2 in the low (except 4.1 mmol/L), medium and high Hbo2 groups were equivalent to those in the non-lipid blood colorimetry (Hbc1), and the two methods were well correlated. The results of light scattering method have nothing to do with the concentration of lipid blood.(4)There was no significant difference of the Hb between the light scattering method and plasma exchange method in three groups ( P>0.05), Both of them have equivalence and good correlation ( R2=0.968,0.948,0.870). Conclusion:Light scattering method can effectively reduce the effect of lipid blood on hemoglobin determination, and can replace the traditional plasma exchange method, which has high clinical application value.

Objective:To investigate the effects of tantalum coating on adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods:In this study, BMSCs were extracted from 6 6-week-old rats and cultured in vitro to the third generation. Tantalum coating was manufactured on Ti6Al4V by chemical vapor deposition. The cells were identified by flow cytometry before they were induced with different mediums for osteogenesis, chondrogenesis and adipogenesis. The adhesion, proliferation and osteogenic differentiation of BMSCs were detected with fluorescence staining, Cell Counting Kit-8 (CCK8) assay and Q-PCR, respectively. Recorded and compared were the adhesion rate, proliferation rate, and expression of osterix (OSX), Runt-related transcription factor 2 (RUNX2), osteonectin (OSN) and osteopontin (OPN) of BMSCs on the surface of titanium alloy round plates (the Ti6Al4V group) and of tantalum coating round plates (the Ta group). Results:The flow cytometry revealed CD44 (94.55%), CD90 (95.01%) and CD34 (0.06%). Alkaline phosphatase (ALP) staining was positive after osteogenic induction for 14 days; Alizarin red staining showed calcified nodules after osteogenic induction for 21 days; oil red O staining was positive after adipogenic induction for 21 days; alcian blue staining found chondrogenic ability after chondrogenic induction for 21 days. Laser confocal microscopy showed that the BMSCs grew in patches aggregated and closely linked on the surface of titanium alloy round plates (in the Ti6Al4V group) and of tantalum coating round plates (in the Ta group). More BMSCs adhered on the tantalum coating plates than on the titanium alloy plates and exhibited better ductility. The proliferation rates of BMSCs on tantalum coating were significantly faster than those on titanium alloy after 1, 3, 5 and 7 days of co-culture in vitro ( P<0.05).Q-PCR showed that tantalum coating promoted the expression of OSN and OPN after 7 days of culture significantly higher than titanium alloy did ( P<0.05).After 21 days of co-culture in vitro, tantalum coating enhanced the expression of OSX, RUNX2, OSN and OPN significantly higher than titanium alloy did ( P<0.05). Conclusion:Compared to titanium alloy which is used for conventional orthopedic implants, tantalum coating can observably promote adhesion, proliferation and osteogenic differentiation of BMSCs.

Objective To investigate the clinical characteristics and angiographic findings of cardiogenic shock(CS)following acute myocardial infarction(AMI) in elderly patients.Methods Between January 2015 and April 2016,we carried out a retrospective observational analysis of consecutive elderly patients in Tianjin Chest Hospital,who suffered CS-complicating AMI.Emergency angiography and percutaneous coronary intervention(PCI) were performed after admission.All selected patients were divided into CS and non-CS groups according to whether CS occurred.Electrocardiograph (ECG),cardiac enzyme testing,and ultrasound cardiography were performed after admission to monitor the occurrence of CS.Results The incidence of CS-complicating AMI was 8.33％ (34/408) in elderly patients.Among all CS patients enrolled,the aged patients accounted for 91.89 ％ (3 4/3 7).In-hospital mortality rate was 2 9.41 ％ (10/3 4).There were significant differences between two groups in WBC,H s-CRP,blood glucose,CR and ALT (t =2.403,4.596,6.778,6.109,each P＜0.05).The NT-Pro BNP level,the time of FMC,the frequency of left main and multivessel disease were higher in the CS group than in the non-CS group (each P ＜ 0.05).Conclusions Elderly patients are bearing high risk of CS following AMI.Prolonged FMC time and the presence of left main and/or multivessel lesion are independent risk factors for the development of CS.The optimal revascularisation strategy can improve the clinical outcome of patients with CS.

This article mainly discussed the concept and application of dynamic neuromuscular stabilization in order to make up for the defect that some of the traditional training only focus on large muscle groups, and ignore the deep trunk muscles, especially the small deep muscles.