BACKGROUND:To build up an effective method of isolating and culturing granule cels is a pivotal step to enhance fertilization-embryo transfer rate. Current studies mainly focus on the isolation methods of human ovarian granulosa cels rather than cel counting, purity and subsequent growth. OBJECTIVE: To establish the effective methods of isolating, purifying and culturing human ovarian granulosa cels in vitro. METHODS: Folicular fluid was harvested from women undergoing fertilization-embryo transfer procedures. Human ovarian granulosa cels were obtained from the folicular fluid by lysis treatment, precipitation method or density gradient centrifugation. Granulosa cel mucus masses were digested with type I colagen enzyme or hyaluronidase and then cultured in the culture medium with or without autologous folicular fluid. RESULTS AND CONCLUSION: Lysis treatment yielded the largest amount of granulosa cels compared to the precipitation method and density gradient centrifugation (P > 0.05,P < 0.05, respectively). Cels prepared by the three methods showed the same cel viability. After 24 hours of culture, the precipitation method obtained the largest amount of adherent granulosa cels (P < 0.05); and the density gradient centrifugation obtained the least amount of cels (P < 0.05). Compared with type I colagen enzyme, hyaluronidase took less time to digest the cels thoroughly. Autologous folicular fluid could promote the growth and survival of granulosa cels. These findings indicate that the precipitation method, though time-consuming, can obtain the highest cel viability and harvested the largest amount of granulosa cels after culture; hyaluronidase is more suitable for digesting granulosa cel mucus mass than type I colagen enzyme; autologous folicular fluid added into the culture medium is more conducive to granulosa cel growth.

Objective To investigate hydrogen peroxide (H2O2) and transforming growth factor-β2 (TGF-β2) induced fibronectin (FN), collagen 1 (COL1), nuclear factor (NF)-κB P65 proteins and interlukin (IL)-1βgene expression in human trabecular meshwork cells (HTMCs), and the interventional mechanism of resveratrol (RSV). Methods (1) HTMCs with 70 to 80%confluency were divided into 5 groups. The experimental groups were treated with serum-free medium and with H2O2 at concentrations of 150, 300, 450 and 800μmol/L. The control group was treated with 0μmol/L H2O2. The protein levels of FN, COL1, NF-κB P65 and NF-κB P65 phosphorylation (P-NF-κB P65) were measured by Western blot assay. The expression of IL-1βgene was measured by qPCR. (2) HTMCs were divided into 3 groups. The control group was treated withserum-free medium and without H2O2 and RSV. The H2O2 group was treated with 300μmol/L H2O2. The H2O2+RSV group was treated with 300μmol/L H2O2 and 25μmol/L resveratrol (RSV). The expressions of proteins and genes mentioned above were detected in three groups. NF-κB P65 nuclear translocation was assessed by immunofluorescence technique. (3) HTMCs were divided into 3 groups. The control group was treated with serum-free medium and without TGF-β2 and RSV. The TGF-β2 group was treated with 5μg/L TGF-β2. The TGF-β2+RSV group was treated with 5μg/L TGF-β2 and 25μmol/L RSV. The expressions of proteins and genes mentioned above were detected in three groups. Results (1) Compared with control group, the protein levels of FN and P-NF-κB P65 were significantly increased in 150, 300, 450 and 800μmol/L groups,the expression levels of COL1 protein and IL-1β gene were significantly increased in 300, 450 and 800 μmol/L groups (P <0.05). There were no statistical significances between other indicators. (2) The expression levels of FN, COL1, P-NF-κB P65 proteins and IL-1βgene were significantly higher in H2O2 group than those in control group, and which were significantly lower in H2O2+RSV group than those in H2O2 group. Compared with control group, only the expression of IL-1βgene was decreased in H2O2+RSV group (P < 0.05). NF-κB P65 was only expressed in cytoplasm in control group, while it was expressed in both cytoplasm and nucleus in H2O2 group. Compared with H2O2 group, NF-κB P65 was mainly expressed in cytoplasm. (3) Compared with control group, the expressions of FN, COL1, P-NF-κB P65 proteins and IL-1β gene were significantly increased in TGF-β2 group (P < 0.05). Compared with TGF-β2 group, the indicators mentioned above were significantly decreased in TGF-β2+RSV group (P<0.05). Conclusion H2O2 and TGF-β2 can upregulate the expression of FN, COL1, P-NF-κB P65 proteins and IL-1βgene in HTMCs, which may be involved in the development and progression of glaucoma. RSV can inhibit the influence of H2O2 and TGF-β2 in HTMCs and exert a protective effect on glaucoma.

Object To study the effect of Haimidin(HMD) on erythrocyte membrane of H 22 ascites tumor bearing mice and DNA synthesis in human gastric tumor cell Methods By fluorescence spectrophotometry and laser scanning confocal microscopy Results HMD can lower erythrocyte microviscosity and elevate membrane lipid fluidity of H 22 ascite tumor bearing mice It also reduced DNA fluorescent intensity and DNA content of gastric tumor cells in vitro Conclusion HMD can improve blood circulation of tumor bearing mice, enhance immune adherence of erythrocyte on tumor cells and displays its antitumor activity by interferring DNA synthesis of tumor cells

Thirty mothers of four dadys later after delivery with agalactia, whose babies needed additional milk over one third of physiological needs, or agalorrhea were chosen to take 125 g of MUYINGLE divided into 3 parts a day for observing its lactigenous effect, and thirty other agalactous mothers, who might chose any kind of traditional Chinese lactagogue foods to eat, as control group. Those subjects whose babie's additional milk was less than one fourth or half of physiolgical needs for agalactia or agalorrhea respectively after four days with MUYINGLE were effectual. The results showed that the lactagogue efficacious rate of MUYINGLE and control group were 86. 7% and 33. 3%, respectively. The lactagogue effects were significant difference between the two groups (P

AIM: To investigate the anti-metastasis effect of weimaining, extracted from fragopyrum cymosum meissn, a Chinese medicine, on murine Lewis lung carcinoma (3LL). METHODS: The anti-metastasis effect of weimaining in vivo was detected in the grafting lung metastasis model of murine Lewis lung carcinoma. The effects of the drug on the expression of CD34 and E-cadherin were investigated by immunohistochemical staining and RT-PCR. RESULTS: Weimaining effectively inhibited the lung metastasis of 3LL at a concentration of 250 mg?kg -1?d -1, significantly suppressed the expression of CD34 and increased the expression levels of E-cadherin protein and mRNA in 3LL cells. CONCLUSIONS: Weimaining inhibits the metastasis of murine Lewis lung carcinoma (3LL) in vivo via increasing the expression of E-cadherin and decreasing microvessel density of tumor tissue.

Objective To knock out Asxl2 gene in murine embryonic fibroblast cell line NIH3T3 using CRISPR/Cas9n system. Methods A pair of sgRNAs which targeted exon 5 of Asxl2 gene were designed and subcloned into the pX462 vec?tor. The recombined plasmids were verified by sequencing and transfected into NIH3T3 cell line. Single cells were isolated through serial dilutions, followed by an expansion period to obtain new monoclonal cell lines. The genomic DNA of the new monoclonal cell lines was extracted and a DNA fragment flanked the target site was amplified by genotyping PCR then se?quenced. Lastly, western blotting were applied to confirm whether Asxl2 was successfully knocked out. Results The CRIS?PR/Cas9n plasmids that targeted Asxl2 were successfully constructed. NIH3T3 cells were co-transfected with the two recom?binant constructs. After puromycin selection, subclonal cell lines were obtained and one of them was validated by genotyping PCR-sequencing. Western blotting also confirmed that Asxl2 was completely depleted in the NIH3T3 cell line. Conclu?sion CRISPR/Cas9n plasmids that targeted Asxl2 were successfully constructed therefore a Asxl2 knockout NIH3T3 stable cell line was established via this system.

Objective: To explore the effect of working memory intervention training on working memory and literacy of children with developmental dyslexia,so as to provide a preference for practice of the trianing of working memory among children with dyslexia. Methods: A total of 32 children with dyslexia of grade 3-5 in a primary school in Guiyang were randomly divided into two groups: the study group (n=16) and the control group (n=16),and the software of training exercises of working memory was applied to conduct interventional trainings of different durations to 2 gruops of children. Results: Through the intervention training of working memory, the scores of literacy and working memory tasks in the study group (2 217.88±252.32, 105.13±7.68) were significantly higher than those in the control group (1 907.69 ± 545.15, 96.50 ± 11.04) (t=2.06, 2.56, P<0.05). Conclusion The working memory ability of children with dyslexia can be improved by working memory intervention training for a certain period of time. The intervention effect is not only significant in the trained working memory task, but also can be extended to other untrained contents such as literacy.

Objective:To investigate the diagnostic values of human epididymis protein 4 (HE4), endothelial cell specific molecule-1 (ESM-1) and epidermal growth factor receptor (EGFR) for lung cancer.Methods:The clinical data of 90 patients with lung cancer and 50 patients with benign lung diseases diagnosed by the pathological examination in Tangshan People's Hospital from December 2019 to January 2021 were retrospectively analyzed, and 40 healthy physical examiners in the same period were selected as the controls. The serum HE4 levels were detected by electrochemiluminescence method. The serum ESM-1 and EGFR levels were tested by enzyme-linked immunosorbent assay. The differences in serum HE4, ESM-1 and EGFR levels between the three groups were compared; logistic regression analysis was used to screen out the effective indicators for the diagnosis of lung cancer and to construct a prediction model for the diagnosis of lung cancer. Using pathological diagnosis result as the gold standard, the receiver operating characteristic (ROC) curve was drawn, and the diagnostic efficacy of indicators for lung cancer was evaluated.Results:The levels of serum HE4 in lung cancer group, benign lung diseases group and healthy control group were 119.55 pmol/L (82.06 pmol/L, 189.00 pmol/L), 58.84 pmol/L (45.62 pmol/L, 69.41 pmol/L) and 42.67 pmol/L (37.09 pmol/L, 51.84 pmol/L), the levels of ESM-1 were 33.00 ng/ml (25.85 ng/ml, 47.40 ng/ml), 20.14 ng/ml (11.93 ng/ml, 28.90 ng/ml) and 15.39 ng/ml (11.84 ng/ml, 20.19 ng/ml), and the levels of EGFR were 46.60 pg/ml (37.45 pg/ml, 58.98 pg/ml), 32.77 pg/ml (26.27 pg/ml, 40.86 pg/ml) and 30.43 pg/ml (27.54 pg/ml, 35.75 pg/ml), and the differences in each indicator among the three groups were statistically significant (all P < 0.001). The levels of serum HE4, ESM-1 and EGFR in lung cancer group were higher than those in benign lung diseases group and healthy control group. In patients with lung cancer, logistic regression analysis was performed with HE4 (X 1), ESM-1 (X 2) and EGFR (X 3) as the independent variables and pathological diagnosis as the dependent variable, and a lung cancer prediction regression model was established: P = 0.171X 1+0.351X 2+0.184X 3-24.660. The accuracy of this model in predicting lung cancer could reach 98.5%, and serum HE4, ESM-1 and EGFR were risk factors for the occurrence of lung cancer (all P < 0.05). The area under ROC curve from high to low was HE4 (0.960), ESM-1 (0.942) and EGFR (0.859). The diagnostic sensitivity of serum HE4 63.67 pmol/L for lung cancer was 86.7%, and the specificity was 97.5%. Both serum HE4 ( r = 0.304, P = 0.004) and ESM-1 ( r = 0.416, P < 0.001) were correlated with EGFR. Conclusions:Serum HE4, ESM-1 and EGFR can be used as effective indicators for the diagnosis of lung cancer, and the prediction model established based on the three serum tumor markers is of good value for the diagnosis and prediction of lung cancer.

Brain-derived neurotrophic factor (BDNF) exerts its survival-promoting effects on photoreceptors and retinal ganglion cells, however, delivery systems with little-to-no side effect are needed to sustain its controlled release and long-term efficacy. Our previous studies demonstrated that adipose-derived stem cells (ADSCs) are ideal delivery systems for gene therapy; moreover, ADSCs present unique properties like migration to damaged tissue sites, immunomodulation and anti-inflammation. Herein, we propose to employ ADSCs as the BDNF gene delivery vehicle. Different Analyses like flow cytometry, differentiation and cell proliferation assays etc demonstrated that BDNF were successfully transduced into ADSCs and the stemness of ADSCs was maintained even with the transduction. Real Time PCR and Western blot were used to measure mRNA and protein expressions of the BDNF-transduced ADSCs. The results demonstrated that the BDNF expression level of the lentiviral-BDNF transduced ADSCs is significantly increased and, moreover, enhanced the expression of other neurotrophic and downstream signaling factors. The data suggest that ADSCs are a reliable delivery vehicle for BDNF and could be used for the treatment of various diseases.

Objective:To evaluate the difference of dosimetry between three-dimensional and two-dimensional plans based on CT images of occult perforation in brachytherapy of cervical cancer, aiming to provide clinical reference.Methods:A total of 817 patients with cervical cancer received simple intrauterine (intrauterine tandem plus vaginal colpostats) three-dimensional brachytherapy in Chongqing University Cancer Hospital from January 2019 to December 2020 were retrospectively reviewed. Among them, 16 patients had occul uterine perforation. Based on Oncentra Brachy Therapy plan system, the single prescription dose was 6Gy. Three-dimensional (3D group) and two-dimensional (2D group) plans were designed on the perforated CT images The target volume, conformal index (CI), conformal index coformity index (COIN) and organs-at-risk (OAR) D 2cm 3 parameters were used to assess the plans between two groups. Results:The incidence of pccult uterine perforation was 1.96%(16/817) during brachytherapy for cervical cancer. The volume of prescription dose curve in the 3D group was (40.74±14.98) cm 3, significantly smaller compared with (91.46±19.71) cm 3 in the 2D group ( P<0.05), whereas the volume of the high-risk clinical target area wrapped by prescription dose curve did not significantly differ between two groups ( P>0.05). The CI and COIN in the 3D group were 0.79±0.10 and 0.72±0.96, significantly higher compared with 0.38±0.09 and 0.37±0.18 in the 2D group (both P<0.05). The D 2cm 3 of bladder, rectum, sigmoid colon, small intestine in the 3D group were (306.06±77.57) cGy, (252.27±72.60) cGy, (127.25±62.84) cGy and (228.79±94.90) cGy, significantly lower than (548.03±164.21) cGy, (411.16±118.74) cGy, (227.45±94.48) cGy and (450.95±157.96) cGy in the 2D group (all P<0.05). Conclusions:Application of image guidance in brachytherapy of cervical cancer is helpful to detect occult uterine perforation. When occult uterine perforation occurs, the use of three-dimensional plan can basically meet the clinical needs, which is significantly better than the two-dimensional plan.