Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D).The highest regeneration rate (34.6%)was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine (BA).The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant (2n = 12)with only one haploid plant(n = 6).Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture orB.chinense.

This study was aimed to establish the method of identifying bupleurum cultivated germplasm using simple sequence repeat (SSR) molecular markers and to initially establish dataset of characteristic SSR bands to the bred cultivars or strains. From the bupleurum SSR primer pairs which were designed in previous work, 50 primer pairs were selected. Two bred strains and 4 other bupleurum cultivated germplasms were used as test materials. Primers pairs were screened with effective PCR amplification and high polymorphism. Meanwhile, conditions for PCR amplification and electrophoresis were optimized. Then, obtained SSR bands were analyzed and a clustering tree on the basis of genetic distance was constructed. The results showed that 9 SSR primer pairs can be used for identification. The suitable assay conditions were established and characteristic SSR bands were obtained for tested materials. The tested samples can be divided into 4 categories in the genetic similarity coefficient of 0.73. TheB. scorzonerifolium cultivated inHeilongjiang andChuanhongchaiNo. 1 strains were clustered as one category. ChuanbeichaiNo. 1 strain andZhongchai No. 1 cultivar clustered as another category. Cultivated germplasms fromSichuan Fengshunand Rongxian clustered as a unique category. It was concluded that the primer pairs and assay method established in the present study can be used as reference in identification of bupleurum cultivars or cultivated germplasms.

In this study, the induction of hairy roots of Bupleurum chinense DC. was explored and established after experiments at different conditions: A. rhizogenes A4 was used to infect the leaves bases of B. chinense tube seedlings. The explants were co-cultured on Phytagel-solidified media for 3 days and then, were turned into solid media, similar with the co-culture media except that bacteriostat was added. After 10 days, rootlets began to appear and after 4 to 5 weeks, rootlets can be converted into liquid shaking culture stage. Plants regeneration from hairy root was useful for the research of new germplasm production and the variety improvement breeding. In the present study, the regenerated plants were obtained. One approach was to continuously culture under light conditions the seedlings which parting off spontaneously from the hairy roots during liquid shaking culture. The other approach was to culture the callus-like tissues produced by hairy roots with the optimized regeneration media for the induction of regenerated plants. The results of present study provide a technique to induce hairy roots and plantlet regeneration of B. chinense and this technique is helpful for the researches on metabolism, especially on the Agrobacterium-mediated genetic transformation of B. chinense.

Aim To explore the effect of genetic poly-morphisms of POR on the stable warfarin maintenance doses in Han Chinese patients receiving mechanical heart valve replacement. Methods The association between POR gene polymorphisms and warfarin doses of 185 Han Chinese patients were investigated through ANOVA or t test. SNPs of POR and VKORC1 were de-tected by Sequenom? DNA MassArray genotyping method. CYP2C9*3 was genotyped by polymerase chain reaction-restriction fragment length polymorphism method ( PCR-RFLP ) . Patients ’ clinical characteris-tics, INR value and daily dose were obtained from their medical records. Statistical analysis was performed by SPSS 21. 0 software. Results No mutant carriers of POR rs17148944 , POR rs56256515 and rs72553971 were found in this study. The genotype frequencies of other SNPs were in accordance with Hardy-Weinberg e-quilibrium. In the group of patients with CYP2C9*1*1 , the mutant type carriers ( T carriers ) of POR rs17685 had a significantly higher dose than CC carri-ers(3. 50 ± 1. 07) mg·d-1 vs (3. 14 ± 0. 94) mg· d-1,P =0. 03. Also, in the group of patients with CYP2 C9*1*1 and VKORC1 rs9934438 G allele carri-ers, the mutant type carriers ( T carriers ) of POR rs17685 had a significantly higher dose than CC carri-ers(4. 76 ± 0. 90) mg·d-1 vs (4. 08 ± 1. 03) mg· d-1 ,P=0. 04. No significant difference was found in different genotypes of POR rs2868177 . Conclusion These results illustrate that POR rs17685 T carrier is closely associated with a higher warfarin maintenance dose, suggesting that this SNP is useful for clinical guidance of warfarin.

Objective To investigate the inhibitory effect of the cytochrome P450 3A4(CYP3A4) gene expression and function in CHL-3A4 cells lines by vector-ecpressed small hairpin interfering RNA(shRNA).Methods The shRNA expression vectors targeting CYP 3A4 gene(CYP3A4Ⅰ,CYP3A4Ⅱ,CYP3A4Ⅲ) were designed and constructed.The inhibitory effect of shRNA was detected by Western blot and RT-PCR analysis.The inhibitory effect of cytotoxic of cyclophosphamide using shRNA was measured by MTT assay.Results CYP3A4Ⅲ shRNA expressing vector significantly reduced the CYP3A4 mRNA(70%) and protein expression levels(75%) of the CYP3A4 gene in CHL3A4 cells suppression of CYP3A4 gene expression by CYP3A4Ⅲ largely reversed cyclophosphamide cytotoxicity(75%) in CHL-3A4 cells.Conclusion Vector-based RNAi could suppress CYP3A4 gene expression and function,and the use of RNAi to inhibit CYP3A4 gene expression in mammalian cells was a promising new tool for the study of cytochrome P450 gene function.

Objective To study the effect and mechanisms of berberine (BBR) on the proliferation of papillary thyroid cancer K1 cells induced by high glucose.Methods K1 cells were cultured under 5.5 mmol/L or 25 mmol/L glucose condition with or without different concentration of BBR (0,10,40 and 80 μmol/L) for 24 hours.The proliferations of K1 cells in each condition were detected by MTT.Western blot was used to measure the expression of nuclear factor erythroid 2-related factor 2 (Nrt2),phosphoinositide 3-kinase (PI3K),protein kinase B (Akt) and phosphorylated-Akt (p-Akt).The distribution pattern of Nrf2 in K1 cells was determined using immunofluorescent staining.Results Compared with 5.5 mmol/L condition,the proliferation rate [(126.64 ± 5.41) ％ vs (87.31 ± 3.67) ％],expression levels of PI3K (0.425 ±0.019 vs 0.272 ±0.039),p-Akt/Akt (0.446 ±0.021 vs 0.168 ±0.035) and Nrf2 (0.597 ± 0.014 vs 0.308 ± 0.026),and Nrf2 distribution (93.0％ vs 23.1％) in nuclear of K 1 cells under 25 mmol/L condition were significantly elevated,respectively (all P ＜0.01).Addition of BBR in 25 mmol/L condition dose dependently (10,40,80 μmol/L) lowered the proliferation rate of K1 cells [(111.76 ± 4.10)％,(70.03 ±2.18)％,(32.41 ±3.76)％ vs (126.64 ±5.41)％,all P＜0.05],and suppressed the expression of PI3K,p-Akt/Akt,Nrf2,and Nrf2 nuclear distribution (P ＜ 0.05).Conclusions BBR dose dependently inhibited the proliferation of high glucose-induced K1 cells.This effect was associated with the suppression on of PI3K/Akt signaling activation,Nrf2 expression and its nuclear translocation.

OBJECTIVETo clone the full-length cDNA of a uridine diphosphate glycosyltransferase (UGT) gene which may be involved in saikosaponin biosynthesis of Bupleurum chinense, and construct the transgenic vectors for over expression and RNAi of the cloned UGT. These works will provide foundation for further its function analysis by transgene study.

METHODRAGE and LD-PCR were used to clone the full-length cDNA of the UGT, on the basis of its partial cDNA sequence obtained from our previous 454-sequenced dataset. The ORF and partial sequences of the UGT were PCR cloned using primers with corresponding restriction enzymes cutting sites. The PCR products were digested with corresponding restriction enzymes and then were inserted into pCAMBIA-SUPER 1 300 and pHANNIBAL. The recombinant pHANNIBAL were digested with Not I and then were inserted into a binary vector, pART27. Finally, the transgenic vectors for over expression and RNAi of the cloned UGT were constructed.

RESULTThe full-length cDNA of a UGT were cloned from B. chinense. The recombinant vectors for over expression and RNAi of the UGT were obtained.

CONCLUSIONOur works on full-length cDNA cloning and transgenic vectors construction provide a substantial foundation for follow-up biofunction analysis of the UGT through transgenic research.

Amino Acid Sequence ; Bupleurum ; genetics ; Cloning, Molecular ; Genetic Vectors ; Glucuronosyltransferase ; chemistry ; genetics ; Molecular Sequence Data ; RNA Interference ; Transgenes

Objective:To investigate the safety of the Triple-P procedure in women complicated with severe placenta accreta spectrum disorders (PAS) and its influence on second pregnancy.Methods:From January 2015 to December 2017, the outcomes of the second pregnancy after the Triple-P procedure in 11 pregnant women complicated with PAS in the Third Affiliated Hospital of Guangzhou Medical University and the First Affiliated Hospital of Zhengzhou University were retrospectively analyzed.Results:By December 2021, a total of 11 pregnant women who underwent the Triple-P procedure for PAS had a second pregnancy, with a median interval of 3 years (2-3 years). Of the 11 pregnant women, 7 delivered after 36 weeks of gestation. The median gestational age was 38 weeks, and 4 terminated within the first trimester. PAS recurred in 1 of 7 pregnant women (1/7) and was associated with placenta previa. All of the 7 pregnant women were delivered by cesarean section, with a median postpartum blood loss of 300 ml (200-450 ml), and only one pregnant woman required blood transfusion. None of the pregnant women were transferred to the intensive care unit, and there were no uterine rupture, bladder injury, puerperal infection, and neonatal adverse outcomes.Conclusion:Pregnant women who underwent the Triple-P procedure for severe PAS could be considered for second pregnancy with strictly management by an experienced multidisciplinary team, which may result in a good outcome.