OBJECTIVE To study the extraction and enrichment technology of chrysosplenides A （CA） and I （CI） in Tibetan medicine Chrysosplenium axillare. METHODS HPLC method was used to determine the contents of CA and CI. The orthogonal experiment was used to optimize the extraction technology of CA and CI in C. axillare using total transfer rate of CA and CI as evaluation indexes， with volume fraction of ethanol， extraction temperature， extraction times and solid-liquid ratio as factors. The validation test was also performed. The enrichment technology of CA and CI in C. axillare was optimized using D101 macroporous adsorption resin as adsorbent， total contents of CA and CI as evaluation indexes， with the volume fraction and dosage of eluent for impurities and target components. The validation test was also performed. RESULTS The optimum extraction conditions of CA and CI from C. axillare were as follows： the medicinal powder of C. axillare was extracted by ultrasound at room temperature for 45 min at one time with 8 times of 50% ethanol. Results of validation tests showed that total transfer rate of CA and CI in C. axillare was 95.43% in average （RSD＝1.02%， n＝3）. The optimal enrichment technology was as follows： the sample solution was added into D101 macroporous adsorption resin column and stood for 1 hour； the impurities were eluted with 20% ethanol 4 BV （column volume）， and CA and CI were eluted with 50% ethanol 4 BV. The results of validation tests showed that total content of CA and CI was 322.7 mg/g in average （RSD＝1.05%， n＝3）， with average enrichment multiple of 11.61 times. CONCLUSIONS The study has successfully optimized the extraction and enrichment technology of CA and CI from C. axillare， and can provide reference for the development and utilization of CA and CI.

Objective:To establish VSV-G qPCR assay for detection of replication competent lentivirus(RCL) and verify its application.Methods:A real-time fluorescent quantitative PCR for VSV-G envelope gene was developed. Several parameters including specificity, linear, amplification efficiency, precision, trueness, dynamic range, limit of detection, limit of quantification and robustness were verified. Preliminary application on CAR-T cells, end of production cells and the harvest of lentivirus vector was performed by using the method developed.Results:The real-time fluorescent quantitative PCR assay for VSV-G was specific for the detection VSV-G without specific amplification on 293T, PBMC and C8166 cells. The linear range of the assay was 1×10 2 copies/test-1×10 9 copies/test with a R2 value more than 0.998 and amplification efficiency between 93% and 98%. The precision (relative standard deviation) of the assay was less than 12% and the trueness (the rate of recovery) of the assay was between 85% and 106%. The limit of detection (LOD) and limit of quantification (LOQ) of the assay was 5 copies/test and 40 copies/test. In addition, the robustness of the assay was also well. All the results of validation illustrated that the assay could meet the detection requirements. All of the 54 samples including CAR-T cells, lentivirus vector and end of product cells after amplification and passage on C8166 cells were negative of RCL by using the established assay. Conclusions:The real-time fluorescent quantitative PCR for VSV-G were established successfully. All of the validation results illustrated that the assay could meet the detection requirements. The application of the assay was conducive to further enhance the safety of the lentivirus vector related products.

Objective:To evaluate the safety, feasibility and operational performance of self-developed medical disposable portable endoscopy (YunSendo) for upper gastrointestinal endoscopy examination in Ba-Ma mini-pigs.Methods:A total of 10 Guangxi Ba-Ma mini-pigs were used in the experiment, and mucosal injury models were established in advance by biopsy forceps in esophagus, stomach, and duodenum. Each experimental animal underwent medical disposable portable endoscopy and Olympus endoscopy (GIF-Q260J) performed by two endoscopists separately. The time when the endoscope reached the duodenum, the number of detected mucosal injuries and endoscopic pictures of different parts with standard image acquisition were recorded. Endoscopic operational performance and endoscopic image quality were evaluated. Different endoscopists recorded experimental results with blind method. The procedures of the two endoscopic examinations were performed by coin-tossing method. The paired t test was used for statistical analysis. Results:There were no statistically significant differences in the insertion time and total operation time between medical disposable portable endoscopy and Olympus endoscopy ( (171.00±9.96) s vs. (164.00±17.84) s, (285.00±33.94) s vs. (273.40±23.46) s; t=1.289 and 1.281, P=0.230 and 0.232). There were no statistically significant differences in the percentage of time of clear visual field during endoscopy insertion and total operation between medical disposable portable endoscopy and Olympus endoscopy ((91.83±1.85)% vs. (91.52±1.51)%, (93.07±3.10)% vs. (92.06±2.57)%; t=0.401 and 0.689, P=0.698 and 0.508). Moreover, there were no statistically significant differences in the score of comprehensive operation performance, score of clear image number, score of image color recognition, score of image illumination, comprehensive score of image quality and number of detected mucosal injuries ((9.66±0.30) points vs. (9.86±0.15) points, (39.50±0.71) points vs. (39.30±1.06) points, (39.70±0.48) points vs. (39.40±0.70) points, (39.40±0.70) points vs. (39.50±0.71) points, (9.88±0.09) points vs. (9.85±0.20) points, 9.80±0.42 vs. 9.90±0.32; t=2.176, 1.000, 1.152, 0.317, 0.629 and 0.557, all P>0.05). There were no adverse events after operation in medical disposable portable endoscopy group and Olympus endoscopy group. Conclusions:The medical disposable portable endoscopy is safe and feasible for endoscopy examination in live animal models. Different parts of upper gastrointestinal tract and mucosal lesions can be clearly detected. The operational performance and the image quality are excellent, which is similar to Olympus endoscopy (GIF-Q260J).

OBJECTIVE: To evaluate the clinical efficacy of weight management combined with pharyngoplasty for treatment of obesity-related obstructive sleep apnea-hypopnea syndrome (OSAHS). METHODS: Sixty obese patients with OSAHS were randomly assigned into the combined treatment group and control group ( RESULTS: After 6 months of treatment, the patients receiving the combined treatment showed significant reductions of BMI, neck circumference and waist circumference as compared with the measurements before treatment and with those in the control group ( CONCLUSIONS Weight management combined with uvulopalatopharyngoplasty can produce a good clinical efficacy for treatment of OSAHS with obesity, and the patients should have strengthened continuous family weight management while receiving surgical treatment.
Body Mass Index ; Humans ; Obesity/surgery* ; Reconstructive Surgical Procedures ; Sleep Apnea, Obstructive/surgery* ; Waist Circumference

To establish a method for isolation, culture and identification of adipose-derived mesenchymal stem cells (ASCs) from the inbreed line miniature pig of Wuzhishan (ILMW).
 Methods: A total of 100 g adipose tissues were obtained from subcutaneous tissues of neck in six-month old healthy ILMW (3 samples, male). ASCs from ILMW (ILMW-ASCs) were isolated from adipose tissues through 0.1% collagenase digestion. The cells at the 3rd, 5th, 8th, 13th passages were collected. Cell morphology, size, phenotype, cell cycle, and apoptosis were monitored. Cell differentiation was induced and cell proliferation curve was drawn.
 Results: The ILMW-ASCs, fibroblast-like or whirlpool-like, began the adherence at 36 h and entered a logarithmic phase in the 5th day. Eighty percent of them were fused in the 7th day. The average diameter and volume of ILMW-ASCs were (17.00±0.54) µm and (2.58±0.24)×10-9 L, respectively. The expressions of CD29, CD44 and CD90 were positive, and there was no significant difference between the different passages (all P>0.05). The expressions of CD45, CD8a and HLA-DR were increased with the increase in passages after the 3th passage (all P<0.05). The adipogenic induction of ILMW-ASCs was observed by positive oil red O staining, and the osteogenic induction of ILMW-ASCs was determined by positive alizarin red staining. Apoptosis and senescence occurred in the 13 passage of ILMW-ASCs, and the proportion of S phase of cell cycle was lower than that in lower passages (all P<0.05). 
 Conclusion: ILMW-ASCs are one of the best choice for porcine ASCs, which might provide a source of candidate stem cells for therapy of large animal disease models and tissue or organ repairment.
Adipose Tissue ; Animals ; Cell Differentiation ; Cells, Cultured ; Male ; Mesenchymal Stem Cells ; Swine ; Swine, Miniature

Objective To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional 51Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy.Methods Four assays including BATDA, CAM (calcein acetoxymethyl ester), CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line.Intra-and inter-assay reproducibility of these assays and their correlation with 51Cr release assay were analyzed.Results After optimization, all of the four cytotoxicity assays showed good correlation with effector to target (E/T) ratio in a certain range.Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1.BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity.Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency.All of the four assays, especially BATDA and CAM assays, showed good intra-and inter-assay reproducibility.Among these assays, BATDA assay showed the best correlation with the traditional 51Cr release assay.BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells.Conclusion Compared with the other three assays, BATDA assay shows the best linear correlation with 51Cr release assay and has the advantages of time-saving and good reproducibility.Besides, it is a better assay for detecting the cytotoxicity of adherent cells.BATDA assay is a promising substitute for 51Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells.

To isolate bone marrow mesenchymal stem cells (BM-MSCs) and establish the model of chronic kidney disease (CKD) of Wuzhishan (WZS) mini-pig, and to study the repairment effect of BM-MSCs on CKD-induced renal fibrosis in vitro.
 Methods: Density gradient method was used to isolate and culture BM-MSCs. The cells were verified by morphology, phenotype, differentiation and so on. The left partial ureteral obstruction (LPUUO) was used to establish the CKD model, which was evaluated by B-ultrasound, single-photon emission computed tomography (SPECT), HE and Masson staining. The cells were divided into 3 groups, the tissue plus BM-MSCs group, the tissue group, and the BM-MSCs group, respectively. Seven days later, the supernatants were collected to observe the changes of hepatocyte growth factor (HGF) cumulative release. HE and Masson staining was used to observe the changes of renal tissue.
 Results: The isolated BM-MSCs possessed the features as follow: fibroblast-like adherent growth; positive in CD29 and CD90 expression while negative in CD45 expression; osteogenic induction and alizarin red staining were positive; alcian blue staining were positive after chondrogenic induction. Twelve weeks after the operation of LPUUO, B-ultrasound showed the thin renal cortical with pelvis effusion; SPETCT showed the left kidney delayed filling and renal impairment. The accumulation of HGF in the tissue plus BM-MSCs group was significantly higher than that in the tissue alone group at the 1st, 5th, 6th, 7th day, respectively (P<0.05). HE staining showed the different degree of renal lesions between the tissue plus BM-MSCs+CKD group and the tissue alone group, which was aggravated with the time going. Masson staining showed that the cumulative optical density of blue-stained collagen fibers in tissue plus BM-MSCs group was significantly lower than that in the tissue group at the 5th to 7th day (P<0.05).
 Conclusion: BM-MSCs from WZS mini-pig can inhibit or delay the progress of CKD-induced renal fibrosis through autocrine HGF in vitro.
Animals ; Autocrine Communication ; physiology ; Bone Marrow Cells ; Cells, Cultured ; Fibrosis ; physiopathology ; prevention & control ; Hepatocyte Growth Factor ; metabolism ; Kidney ; drug effects ; pathology ; physiopathology ; Mesenchymal Stem Cells ; drug effects ; Renal Insufficiency, Chronic ; complications ; physiopathology ; Swine ; Swine, Miniature ; Ureteral Obstruction ; complications

Abstract] Objective To establish an assay for the detection of porcine parvovirus ( PPV) and to verify its application for monitoring cells used for production.Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1).Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated.A stock of PPV strains was prepared by infecting swine testicle ( ST) cells with PPV strains.An assay for the detection of PPV infection was developed by using ST cells as sensitive cells.A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay.The sensitivity of ST cell infection-PCR test was analyzed.The cell samples used for production of biological products were detected by using the established assay.Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses.The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98.The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl.Both of the intra-and inter-coefficient of variation (CV) were less than 5%in Ct values.The intra-and inter-CV in copies of detection were 5%-15% and 30%-40% respectively.The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml.The PPV strains were detected in cell samples with no interference.The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml.All of the 22 cell samples were negative for PPV by using the real-time fluorescent quanti-tative PCR assay.Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully.The application of the two assays was conducive to further enhance the safety of using cells for production and therapy.

Objective To establish an assay for the detection of Torque teno sus virus ( TTSuV) strains and to analyze its preliminary application to biologics.Methods Primers and probe were designed according to the conserved sequences.A fluorescent PCR assay for the detection of TTSuV strains was estab-lished.Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified.The fluorescent PCR assay was performed to detect the samples of por-cine blood, cell substrate and rotavirus vaccine.The porcine blood samples positive for TTSuV strains were further genotyped.Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detec-ted.The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 cop-ies/μl for TTSuV2 genotype with a R2 value more than 0.993.The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype.The intra-and inter-CVs were both less than 7%in Ct values and less than 25% and 45% respectively in copies.No interfer-ence was found in the detection of TTSuV nucleic acids from cell samples.8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes.Com-pared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99%and 98%homologies in sequences.All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains.Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics.

Objective To investigate the risk factors of incidence of hypertensive disorder complicating pregnancy(HDCP)and provide reference for prevention and treatment of HDCP.Methods The clinical information of 2 217 cases of maternal delivery were retrospectively analyzed;The relevant social factors and obstetric factors of HDCP were analyzed.Results 186 cases of HDCP occurred in 2 217 cases of maternal,the incidence rate of HDCP was 8.4%.The single-factor analysis showed that the place of residence.prenatal system checks and parity had certain degree of contact with incidence of HDCP(χ2=11.2331,10.6781,5.4456,all P＜0.05),and the age,abortion history and parity had no significant correlation with incidence of HDCP(χ2=4.9201,3.0134,1.7301,all P＞0.05).Multivariate Logistic regression analysis showed that the rural living and no prenatal system check were risk factors of incidence of HDCP.Conclusion The maternal of rural living and no prenatal system check were the high-risk population of HDCP,and emphasis assessment and reasonable control these factors could reduce the incidence of HDCP.