In order to investigate the genetic basis of morphological variation of tetraploid plantlets of Atractylodes macrocephala, diploid plantlets were taken as experimental material, sterile filtration colchicine was used to soak 0.5-1.0 cm long buds. The difference between morphology and stomatal of diploid and tetraploid of A. macrocephala was compared, and genome polymorphism was explored by AFLP. The results showed that the buds dipped in 0.1% colchicine solution for 36 h was optimal conditions to induce tetraploid of A. macrocephala with induction rate of 32.0%. Morphological indexes such as leaf area index, leaf length and width, the density of stomas and the number of chloroplast of tetraploid were distinctly different from diploid. Four hundred and fifty-one bands ranging with 80-500 bp were amplified with 24 pairs of primers, the rate of polymorphism was 32.59%. These amplification sites of diploid were different from tetraploid of A. macrocephala, and the differences in morphology of them were reflected in the DNA polymorphism.
Amplified Fragment Length Polymorphism Analysis ; methods ; Atractylodes ; genetics ; Sequence Analysis, DNA ; Tetraploidy

The proliferating cell nuclear antigen (PCNA) gene expression was blocked and retinal pigment epithelium (RPE) proliferation was inhibited by using antisense oligonucleotides (AS-ODN) mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy (PVR). RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN (0.07, 0.28 and 1.12 micro mol/L and sense oligonucleotides (S-ODN with the same concentrations as AS-ODN) mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by 3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression of PCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density (AOD) was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN (0.28 and 1.12 micro mol/L) markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant (P < 0.05 and P < 0.01, repectively) as compared with blank control group. AOD was well correlated with CPM (r = 0.975). It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

Objective　To compare the changes in the genetic diversity of Anopheles minimus through the research on the population genetic characteristics of Anopheles minimus between different years in Nabang Town, Yingjiang County, Yunnan Province. Methods Anopheles mosquitoes were collected by light traps in Nabang Town, Yingjiang County, Yunnan Province in May 2014 and May 2021. After morphological identification, each mosquito was individually stored in separate tubes for further analysis. DNA of Anopheles minimus was extracted using kits. Microsatellite sequences in the template DNA were amplified using eight pairs of fluorescent primers, and the resulting products were subjected to capillary electrophoresis by a sequencing company. PopGen32 software was used to calculate the observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and Shannon's information index (I) for individual microsatellite loci and population groups. PIC-CALC software was used to calculate the polymorphic information content (PIC). Results A total of 158 mosquitoes belonging to 6 Anopheles species were captured in 2014, while 529 mosquitoes belonging to 5 Anopheles species were captured in 2021. The composition ratio of Anopheles minimus among the mosquito species differed significantly between 2014 and 2021 (χ2=70.48, P<0.01). For 8 microsatellite loci, a total of 85 alleles were detected, a range of 6-20 alleles per locus and an average of 10.625 alleles. Ne ranged from 1.717 to 7.797, with an average of 4.011. The highest PIC was found in the am4 locus, and the lowest in the am25 locus. For the population groups, 77 alleles were found in 2014, and 62 alleles were found in 2021. Ne ranged from 1.630 to 8.658, with an average of 4.147 in 2014. Ne ranged from 1.760 to 6.744, with an average of 3.698 in 2021. The average Ho was 0.641 in 2014 and 0.650 in 2021, while the average He was 0.699 in 2014 and 0.691 in 2021. The Shannon's index ranged from 0.774 to 2.493 in 2014, with an average of 1.579, and from 0.938 to 2.224 in 2021, with an average of 1.464. Na, Ne, I, and PIC were all higher in 2014 compared to 2021, with Na: 9.625>7.750, Ne: 4.147>3.698, I: 1.579>1.464, and PIC: 0.655>0.640, respectively. Conclusions The populations of Anopheles minimus in Nabang Town, Yingjiang County, exhibited high levels of polymorphism in both 2014 and 2021. However, the genetic diversity of the population in 2021 was lower than that in 2014.

Objective To comprehensively study the oxidative stress of bone tissue in rats with chronic fluorosis treated with anti-oxidant,the oxidative damage of lipid,protein and DNA.Methods Forty Wistar rats weaned 2 weeks were randomized by weight and divided into 4 groups according to body weight,control group (treated with tap water) and 3 NaF (sodium fluoride) exposure groups (treated with NaF at 50,150 and 250 mg/L),5 female rats and 5 male rats in each group.NaF was given through drinking water.After 6 months of treatment,a 12-hour urine samples were collected,then rats were killed,serum was collected,right rear tibiofibula was separated.Bone and urinary fluoride content and incidence rate of dental fluorine were studied and the levels of bone tissue suppression function of hydroxy free radical,superoxide dismutase (SOD),catalase (CAT),glutathione peroxidase (GSH-Px),8-hydroxydeoxyguanosine (8-OHdG),protein carbonyls (PCO),and malonaldehyde (MDA) were assayed.Results ① Results of suppression function of hydroxy free radical:The difference of bone tissue suppression function of hydroxy free radical among control [(22.99 ± 4.31)U/mg prot],low-excess dose [(22.76 ± 8.11)U/mg prot],medium-excess dose [(13.47 ± 4.56)U/mg prot] and high-excess dose [(19.40 ± 5.92)U/mg prot] groups was statistically significant (F =5.01,P ＜0.05).②Results of SOD:The difference of bone tissue SOD among control [(5.06 ± 1.16)U/mg prot],low-excess dose [(5.32 ± 1.18)U/mg prot],medium-excess dose [(3.71 ± 0.72)U/mg prot] and high-excess dose [(4.80 ± 1.10)U/mg prot] groups was statistically significant (F =4.44,P ＜0.05).③ Results of CAT:The difference of bone tissue CAT among control [(25.20 ± 5.91)U/mg prot],low-excess dose [(22.53 ± 7.10) U/mg prot],medium-excess dose [(17.96 ± 4.71)U/mg prot] and high-excess dose [(19.52 ± 5.52)U/ mg prot] groups was statistically significant (F =2.85,P ＜0.05).④Results of GSH-Px:The differences of bone tissue GSH-Px among control [(52.86 ± 12.88)U/mg prot],low-excess dose [(70.05 ± 15.72)U/mg prot],medium-excess dose [(51.55 ± 6.97)U/mg prot] and high-excess dose [(57.47 ± 10.99) U/mg prot] groups was statistically significant (F =4.89,P ＜0.05).⑤Results of PCO:The differences of bone tissue PCO among control [(58.73 ± 20.86)ng/L],low-excess dose [(89.41 ± 26.20)ng/L],medium-excess dose [(97.07 ± 22.24)ng/L] and highexcess dose [(83.96 ± 29.55)ng/L] groups was statistically significant (F =4.43,P ＜0.05).⑥Results of 8-OHdG:The differences of bone tissue 8-OHdG among control [(87.66 ± 6.32)ng/L],low-excess dose [(86.31± 6.30)ng/L],medium-excess dose [(92.17 ± 4.28)ng/L] and high-excess dose [(88.02 ± 6.14)ng/L] groups was not statistically significant (F =1.88,P ＞ 0.05).⑦Results of MDA:The differences of bone tissue MDA among control [(3.70 ± 1.73) nmol/mg prot],low-excess dose [(2.10 ± 0.95)nmol/mg prot],medium-excess dose [(3.32± 2.20)nmol/mg prot] and high-excess dose [(2.71 ± 2.18)nmol/mg prot] groups was not statistically significant (F =1.37,P ＞ 0.05).Conclusions The activity of SOD and CAT of bone tissue are inhibited and suppression function of hydroxy free radical is decreasing under fluorosis influence,which results in protein damage.Oxidative stress is considered to be one of the mechanisms of skeletal fluorosis.

0.05).The blood loss in harmonic scalpel group was less than that in titanium clamp group or conventional group(P=0.001).CONCLUSION Harmonic scalpel is a new type of surgical instrument and is suitable for endoscopic thyroid surgery.It is worth of recommending that harmonic scalpel can not only increase safety of surgery and reduce blood loss,but also decrease the difficulty of operation and shorten the operation time.

OBJECTIVETo establish and evaluate a BA-ELISA method for the quantitative detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein.

METHODSWe deliberately selected three tables of CFTR and made the synthetic peptide be expressed in E. coli, then used the antigen to immunize rabbits to obtain the anti-CFTR polyclonal serum. After that, 96 well plates were coated with the purified antibody against CFTR. The antigen CFTR which was extracted from human sperm was detected by anti-CFTR antibody labeled with biotin, horseradish peroxidase conjugated avidin, and the substrate. The concentrations of two kinds of antibodies and the experiment parameters were optimized. Thereby, the double antibody sandwich BA-ELISA method for the quantitative detection of CFTR protein was established. Furthermore, the reproducibility, specificity and so on were evaluated by clinical specimens of sperm.

RESULTSThe optimal concentration of coated anti-CFTR IgG was 4 µg/ml, while the biotin labeled anti-CFTR IgG was 10 µg/ml; the optimal blocking buffer was 1% BSA-PBST, the optimal time of the reaction between antigen and antibody was 60 min, the optimal chromogenic time was 15 min, the intra-assay and inter-assay coefficient were 2.16%-9.23% and 2.29%-11.71% respectively; The lowest detectable limit was 0.15 ng/ml; the standard curve had a good linear correlation of R2 = 0.962.

CONCLUSIONThe BA-ELISA method for the quantitative detection of CTFR protein is successfully established, and it is demonstrated that the method has strong specificity, high sensitivity and good reproducibility. It provides the basis and evidence of the further application of the method.

Animals ; Antibodies ; Cystic Fibrosis Transmembrane Conductance Regulator ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; Humans ; Peptides ; Rabbits ; Reproducibility of Results ; Sensitivity and Specificity

Objective To explore the developmental characteristics of different functional levels of autism and the diffe-rence in children. Methods A total of 159 autistic children aged 13-71 months including 85 high-functioning autistic (HFA) chil-dren and 74 low-functioning autistic (LFA) children were evaluated for their developmental characteristics by the development scales from the psycho-educational profile-revised (PEP-R). Results Within the same age group, both HFA and LFA children had obvious unbalance in individual ability development (H=41.68~113.51, P<0.01). Impaired cognitive performance is most common in HFA children. The development of cognitive performance was inconsistent with cognitive expression while the gross motor development was normal. The ability development in LFA children were all impaired, especially in imitation, cogni-tive performance and cognitive expression. With increasing age, the scores of imitation, perception, cognitive performance, cog-nitive expression and overall development quotient were increased gradually in HFA children (P<0.05) while the scores of cogni-tive performance and overall development quotient were decreased in LFA children. Conclusions Autistic children tend to have abnormal developmental progress and order, and individual ability development varies by functional level in autistic children with increasing age.

Objective: To observe the clinical efficacy of acupuncture plus navel acupuncture for patients with urinary retention after radical hysterectomy for cervical cancer. Methods: A total of 64 patients with urinary retention after radical hysterectomy for cervical cancer was divided into a navel acupuncture group (22 cases), an acupuncture group (18 cases) and an acupuncture plus navel acupuncture group (24 cases). All three groups received bladder function training and neuromuscular electrical stimulation. In addition, navel points were combined in the navel acupuncture group. Electroacupuncture was conducted to Qihai (CV 6), Zhongji (CV 3), Dahe (KI 12), Shuidao (ST 28), Ciliao (BL 32) and Huiyang (BL 35) in the acupuncture group. The acupuncture plus navel acupuncture group received both treatments. The catheter was removed after 3 d of treatment. Spontaneous urination, residual urine volume, urinary catheter dependence and recurrence after 3 d, 6 d and 9 d of treatment in each group were observed, respectively. Results: In the acupuncture plus navel acupuncture group, the markedly effective rates after 3 d, 6 d and 9 d of treatment were significantly higher than those in the navel acupuncture group and the acupuncture group; the urinary catheter dependence was lower than that of the other two groups, and the differences were statistically significant (P<0.05, P<0.01); the spontaneous urination time was shorter than that of the navel acupuncture group and the acupuncture group, and the differences were statistically significant (P<0.05, P<0.01); the residual urine volume was significantly less than that of the navel acupuncture group and the acupuncture group, and the differences were statistically significant (both P<0.01). After the catheter was removed, recurrence was observed from the next day after spontaneous urination was resumed. There were 2 cases of recurrence in the navel acupuncture group, 2 cases in the acupuncture group and 1 case in the acupuncture plus navel acupuncture group. The recurrence rate of the acupuncture plus navel acupuncture group was significantly lower than that of the navel acupuncture group and the acupuncture group (both P<0.01). Conclusion: Acupuncture plus navel acupuncture has satisfactory efficacy for urinary retention after radical hysterectomy for cervical cancer. It can significantly shorten the urinary retention time, reduce the patient's dependence on urinary catheter, and reduce the residual urine volume.

Objective To screen the splicing isoforms of estrogen receptor βin the Beagle hypothalamic -pituitary -gonadal axis.Methods For ERβmRNA CDS sequence of eight exons, primers were designed confined to the CDS sequences of two sequential exons.Beagle hypothalamus, pituitary, ovary and uterus tissue cDNA were used as template, and corresponding sequences were amplified by PCR.PCR products were sequenced and aligned in the NCBI web site.The correct gene was then analyzed with DNAMAN comparative analysis software and handwork checking up, thus got the ERβsplicing isoforms of Beagle. Results Four beagle ER beta splicing isomers were obtained:exon 4 complete skipping ER βisomer (300 bp missing), two kind of Beagle ERβisoforms with partial exon 4 and partial exon 5 complicated missing (isoformⅠ334 bp missing and isoformⅡ265 bp missing), and exon 7 complete missing ERβsplicing isoforms (181 bp missing).Exon 4 complete skipping and exon 7 complete missing isomers had been obtained full length coding sequence, and the other two splicing isomers were partial coding sequence.Conclusion This project gained four ERβsplicing isomers of Beagle, and that will lay an important foundation for further study of their roles in the Beagle reproductive regulation mechanism.

Abstract: Objective　To understand the kdr (knockdown resistance, kdr) gene mutation of the voltage-gated sodium channel (VGSC) of Anopheles sinensis in Yunnan Province. Methods　From 2018 to 2019, mosquitoes were collected in Luoping County, Suijiang County, Tengchong City, Yingjiang County, Yuanjiang County and Mengla County in Yunnan Province. The collected mosquitoes were morphologically identified as Anopheles sinensis and genomic DNA was extracted by kits. The DNA templates were sequenced after PCR amplification and the sequencing results were identify as Anopheles sinensis by homology alignment in NCBI. After the ⅡS5 and ⅡS6 fragments of the sodium channels in Anopheles sinensis were amplified and sequenced, the sequencing results were multiple aligned by DNAMAN software, and the mutations were analyzed one by one with BioEdit software to determine the kdr allele types and genotypes, and the frequencies were calculated. Results This survey amplified 287 sequences, and the sequence maps showed that 1014 loci had three alleles, including wild type TTG/L (89.20%), mutant type TTT/F (9.76%) and TCG/S (1.04%). Five genotypes: homozygous wildtype L/L (85.02%), homozygous mutant F/F (6.27%) and S/S (0.35%), heterozygous mutant L/F (6.97%) and L/S (1.39%). The wild type allele TTG/L was the main allele in six sampling sites except Suijiang County. The frequency of wild type allele in Tengchong City was the highest (100.00%). That is, no mutation was detected, while the rest of counties occurred different degrees of mutation at 1014 loci. The frequency of mutant allele in Suijiang County was the highest, reaching 55.68%. Luoping County, Mengla County and Suijiang County had two mutant types. Yingjiang County and Yuanjiang County had one heterozygous mutant L/F. Conclusion　Wild type L1014 (TTG/L) is still dominant in most areas of Yunnan Province. The kdr mutation type is mainly L1014F, followed by L1014S, and the mutation frequency is lower than that in central provinces of China.