Objective To explore the cultural method of oral keratinocytes and make preparations for further investigation in using oral keratinocytes as a new choice of seed cells for the reconstruction of tissue-engineered urethra. Methods Oral keratinocytes of rabbits were isolated in vitro and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3) or a culture dish without i3T3 respectively. Cell morphology was observed and cell growth was detected at intervals.Meanwhile,oral keratinocytes obtained from in vitro culture were performed immunofluorescence staining with broad-spectrum keratin antibody(AE1/AE3) and keratin 19 antibody(K19).The percentage of positive cells of passage 2 reactive to AE1/AE3 was assessed by flow cytometry. ResultsOral keratinocytes seeded onto a feeder layer of i3T3 exhibited finer morphous,better amplification capability,and could be passed for 7 or 8 generations.However,those cultured without i3T3 took on various morphous and could only be subcultured 2 generations before ageing.It was indicated by immunofluorescence staining that oral keratinocytes obtained from in vitro culture were positive for AE1/AE3 staining and 40% were positive for K19 staining.The result of flow cytometry revealed that the amout of positive keratinocytes reactive to AE1/AE3 was more than 95% of total cellular score. Conclusion Oral keratinocytes of rabbits can be cocultured with i3T3 in vitro and magnitude quantity can be attained,laying a favourable foundation for oral keratinocytes as a new choice of seed cells for urethral reconstruction with tissue engineering.

Objective To understand the current situation of endemic fluorosis in Shandong Province,and to provide a scientific basis for development of control strategies.Methods In accordance with the requirements of the national Surveillance Scheme of Drinking-Water-Borne Endemic Fluorosis (Trial) and Shandong Provincial Surveillance Scheme of Drinking-Water-Borne Endemic Fluorosis (Trial),and to understand the progress of the province's water-improvement projects,10 counties were chosen in the province,and 10 water-improvement projects were selected in each county.Running condition of the water-improvement projects was investigated,and water fluoride content was tested.Three epidemic villages were chosen as fixed monitoring villages in each county.In each village,fluoride content in drinking water was determined,and dental fluorosis of all children aged 8 to 12 was diagnosed.Water fluoride content was determined by fluoride ion selective electrode method,and dental fluorosis of children aged 8 to 12 was diagnosed by Dean method.Results ① There were 3570 water defluoridation projects in the province,and water-improvement rate was 87.49％ (10 122/11 569).Normal operating projects accounted for 89.10％ (3181/3570),and the passing rate of water fluoride content was 73.14％ (2611/3570),with the maximum water fluoride as 9.71 mg/L.② In the 10 counties,a total of 86 waterimprovement projects were monitored.Normal operation,intermittent operation and scrapped projects accounted for 95.35％ (82/86),3.49％ (3/86) and 1.16％ (1/86),respectively,and passing rate of fluoride content in water was 50.00％ (43/86),and the maximum water fluoride was 5.32 mg/L.③ In the 27 monitored villages with improved water quality,the passing rate of fluoride content in water was 74.07％ (20/27),and the maximum water fluoride was 4.50mg/L.In the 3 monitored villages without improved water quality,villages of mean water fluoride ≤ 1.20 mg/L and ＞ 1.20 mg/L accounted for 33.33％ (1/3) and 66.67％ (2/3),respectively,and the maximum water fluoride was 1.53 mg/L.④ In the 27 monitored villages with improved water quality,the detection rate of dental fluorosis among children aged 8 to 12 was 57.65％(791/1372),and the detection rate of defect type dental fluorosis was 7.80％(107/1372); the index of dental fluorosis was 1.15.In the 3 monitored villages without improved water quality,the detection rate of dental fluorosis among children aged 8 to 12 was 50.97％ (80/155),and the detection rate of defect type dental fluorosis was 7.10％(11/155); the index of dental fluorosis was 1.16.Conclusions In Shandong Province,the running condition of water improvement project to reduce fluoride still needs to be further improved,and the water fluoride is seriously overweight and endemic fluorosis has not been effectively controlled.Preventive measures should be strengthened.

OBJECTIVETo explore the efficacy differences in early intervention of different acupuncture and moxibustion methods on gastrocnemius fatigue in rats induced by electrical stimulation.

METHODSFifty male SD rats were randomly divided into a control group, a model group, a hand acupuncture group, an electroacupuncture group and a moxibustion group, 10 rats in each group. Electrical stimulation of the sciatic nerve was given in the control group and gastrocnemius fatigue test was induced by electrical stimulation of the sciatic nerve in the model group after anesthesia without other treatment, but just take 6 times interval stimulation in the control group. The hand acupuncture group, the electroacupuncture group and the moxibustion group were treated with the corresponding acupoints stimulation method respectively for 20 min before gastrocnemius fatigue test, and Dazhui (GV 14) and Zusanli (ST 36) were selected. Immediately after gastrocnemius fatigue test, three or four gastrocnemius tissues at the same site on the right side were quickly taken for making specimen for transmission electron microscope (TEM). The changes of skeletal muscle ultrastructure of myofibrils, mitochondria, sarcoplasmic reticulum, glycogen particles were observed under TEM.

RESULTS(1) Muscle fibers disorder, partial mitochondrial vacuolization and glycogen particles smaller were shown in the model group. (2) No abnormalities were shown in the hand acupuncture group and the moxibustion group with mitochondrial morphology and number, which better than that in the model group, and glycogen particles increased. (3) Abnormal changes in morphology were shown in the electroacupuncture group with part of the muscle fibers derangement, Z line malalignment and a few mitochondria vacuolization.

CONCLUSIONHand acupuncture, electroacupuncture and moxibustion have the different effects on ultrastructure of gastrocoemius in rats. Acupuncture and moxibustion have shown good effects on the prevention and treatment of exercise-induced skeletal muscle cell and organelle damage and delaying exercise-induced fatigue.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Glycogen ; metabolism ; Male ; Mitochondria ; metabolism ; ultrastructure ; Moxibustion ; Muscle, Skeletal ; metabolism ; ultrastructure ; Rats ; Rats, Sprague-Dawley

Objective To investigate the current status of endemic fluorosis in Shandong province,and to provide a scientific basis for the development of preventive strategies.Methods In accordance with the requirements of the national Technical Implementation Plan for Endcmic Disease Control Project in 2009 and Drinking-Water-Borne Endemic Fluorosis Monitoring Program (trial),ten counties were selected to carry out the epidemiological investigation,to understand the progress and the effect of the water improvement projects; three villages were selected in each county,to determine fluoride content of drinking water,to check dental fluorosis of children aged 8 to 12 and skeletal fluorosis of adults over the age of 25.Water fluoride content was determined by fluoride ion selective electrode method,children dental fluorosis was diagnosed with Dean method and adults skeletal fluorosis by the National Standard for Diagnosis of Endemic Skeletal Fluorosis (WS 192-2008).Results There were a total of 687 improved-water defluoridation projects of the 10 counties,the improved-water rate was 83.34％ (3247/3896).The investigation was taken place in 84 improved-water projects of the 10 counties,the projects running normally accounted for 98.81％ (83/84); 41 projects had water fluoride concentration ≤1.20 mg/L,and the pass rate was 48.81％(41/84),with the maximum water fluoride as 5.76 mg/L.In the 26 villages with improved-water projects of the 10 counties,villages with a mean water fluoride ≤ 1.20 mg/L were 15,which accounted for 57.69％ (15/26),＞ 1.20 mg/L were 11,which accounted for 42.31％ (11/26),with the maximum water fluoride as 5.58 mg/L.In the 4 yet to improve water quality villages of the 10 counties,1 village had mean water fluoride ≤ 1.20 mg/L,3 villages had mean water fluoride ＞ 1.20 mg/L,with the maximum water fluoride as 2.92 mg/L.A total of 1331 children aged 8 to 12 were checked in the 26 improved water villages,the detection rate of dental fluorosis was 59.73％ (795/1331),the index of dental fluorosis was 1.17 and the defect rate was 10.14％ (123/1331).A total of 138 children aged 8 to 12 were checked in the 4 yet to improve water quality villages,the detection rate of dental fluorosis was 51.45％(71/138),the index of dental fluorosis was 0.95 and the defect rate was 0.72％ (1/138).In water quality improved villages and yet to improve water quality villages,X-ray detection rates of skeletal fluorosis were 8.80％ (113/1284) and 3.05％ (6/197),respectively,in adults over the age of 25.Conclusions The water fluoride content of improved-water defluoridation projects exceeds the national standard seriously and fluorosis has not been effectively controlled in Shandong province.

Objective　To evaluate the technique and clinical outcomes of modified transperitoneal laparoscopic radical prostatectomy.　Methods　A total of 285 patients received the operation with mean age of 67 years (50-76 years) from January 2008 to April 2012.Mean level of PSA was 15.7 μg/L (1.8 -50.0 μg/L),and mean prostatic volume was 44 ml (26 -74 ml). No lymph node or seminal vesicle involvement was found by CT or MR and radionuclide bone scan revealed no metastasis.271 cases were confirmed diagnosis by prostatic biopsy and 14 were detected through pathological studies of TURP specimens.Gleason score ranged from 6 to 8.14 cases were in clinical stage T1b,29 cases in T1c,214 cases in T2 and 28 cases in T3a.Transperitoneal approach and modified technique involving bladder neck dissection,nervesparing technique and vesicoureteral anastomosis were applied on patients.　Results　Mean operative time was 105 min (55 -150 min).Mean intraoperative estimated blood loss was 240 ml (50-800 ml).Rectal injures occurred in 2 cases and were repaired under laparoscopy.Drainage tube and urinary catheter were removed 48 -72 h and 5 -8 d postoperatively.Postoperative hospital stay was 7 d (5 - 11 d).Positive surgical margin was present in 58 patients.Mean follow-up time was 29 months (3 -50 months).Complete continence were found in 208 patients immediately after catheter removal.68 patient recovered continence within 3 months and 9 patients remained incontinence 3 months after surgery. Normal erection presented in 42 of the 57 cases with nerve-sparing.　Conclusions　Transperitoneal laparoscopic radical prostatectomy is safe and efficient.Higher efficiency and lower complication rate have been achieved through modified laparoscopic technique involving bladder neck dissection,nerve-sparing technique and vesicoureteral anastomosis.

A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).
Acyltransferases ; genetics ; isolation & purification ; metabolism ; Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Huperzia ; enzymology ; genetics ; Molecular Sequence Data ; Plant Leaves ; enzymology ; genetics ; Plants, Medicinal ; enzymology ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Sequence Alignment ; Substrate Specificity

Objective To explore the current profile of drowning related high risk behaviors among the floating children inNingbo City and to identify the risk factors on these behaviors.Methods A total of 7 600 students from grade 1 -9 ineight urban migrant workers'children schools were recruited and surveyed by the questionnaires.And the logistic regressionmodel was used for the analysis of risk factors.Results In last one year,without adult supervision,the incidence rate ofdrowning related high risk behaviors was 27.53%.The results of multivariate logistic regression showed that males (OR =2.30,95%CI:1.99 ~2.65),senior grade (OR =1.23,95%CI:1.18 ~1.27),other juvenile companion on the way toschools (OR =1.26,95%CI:1.06 ~1.51),being able to swim (OR =2.09,95%CI:1.77 ~2.46)and there being theopen water around school and home (OR =1.75,95%CI:1.52 ~2.00)could increase the incidence of drowning relatedhigh risk behaviors.And higher awareness of drowning prevention (OR =0.99,95%CI:0.98 ~0.99),higher rate ofcorrect attitude (OR =0.99,95%CI:0.98 ~0.99),getting along well with schoolmates (OR =0.69,95%CI:0.51 ~0.95)and with family members (OR =0.33,95%CI:0.24 ~0.46)could reduce the incidence of drowning related highrisk behaviors.Conclusion The incidence rate of drowning related high risk behaviors was high among the floatingchildren in Ningbo City,and males,being able to swim might increase the occurrence of high risk behaviors.

Objective To learn the epidemic characteristics and time trends of drowning-induced mortality among the Ningbo residents aged less than 20 years and to provide the scientific evidence for developing drowning prevention strategies. Methods Data were obtained from the death registry system in Ningbo City during 2002-2015 for descriptive analysis, and a linear regression model on an absolute scale or a log scale of the relevant indexes was mainly used to identify the time trends. Results There were all 982 deaths owning to drowning among the Ningbo residents aged under 20 years, with the average crude mortality of 6.23/100000. And the average crude drowning-induced mortality was 8.74/100000, 3.59/100000, 3.85/100000 and 7.64/100000 for the male, female, urban and rural respectively. The standardized mortality rate for drowning showed a significant decreasing trend with the Annual Percent Change (APC) of -9.71% (P<0.001) . APC for the male was -10.03% (P<0.001), higher than -9.64% for the female (P=0.001) . Meanwhile, APC for the urban was-14.51% (P<0.001), also higher than -8.71% for the rural (P<0.001) . The most deaths occurred in the nature water body (90.04%) . Conclusion Though the drowning-induced mortality among the Ningbo residents aged less than 20 years revealed a significance of the decreasing trends, and drowning still emerged as a serious public health issue. Gender parity and regional disparities should take into account when developing some intervention strategies.

Objective:To investigate the effect of TLK2 expression regulated by miR-21 on proliferation and apoptosis of acute myeloid leukemia cells.Methods:Seventy patients with AML admitted to our hospital from January 2019 to July 2022 were selected,while 30 patients with iron deficiency anemia were selected as the control group.Bone marrow mononuclear cells(BMMNCs)of the patients were obtained using Ficoll density gradient centrifugation.RT-qPCR was used to determine the expression levels of miR-21 and TLK2 mRNA in BMMNCs.Mimics-miR-21,mimics-NC,inhibitor-miR-21,inhibitor-NC and NC were transfected into HL-60 cells using liposome-mediated transfection technology.CCK-8 method was used to determine the activity of transfected HL-60 cells after treatment with cytarabine.The apoptosis rate of HL-60 transfected cells was determined by TUNEL method.The expression of TLK2 mRNA in HL-60 cells transfected with inhibitor-miR-21 was determined by RT-qPCR.Results:The relative expression levels of miR-21 and TLK2 mRNA in BMMNCs of AML patients were significantly higher than those of controls(both P＜0.05).After HL-60 cells were treated with cytarabine,both the cell activity of inhibitor-miR-21 group and mimics-miR-21 group decreased significantly with the increase of cytarabine concentration(both P＜0.05).However,at each concentration point of cytarabine,the cell activity of inhibitor-miR-21 group was lower than that of control group(P＜0.05),while mimics-miR-21 group was higher than control group(P＜0.05).After HL-60 cells were treated with cytarabine,the apoptosis rate of inhibitor-miR-21 group was significantly increased(P＜0.05),while that of mimics-miR-21 group was significantly decreased(P＜0.05).After HL-60 cells were treated with inhibitor-miR-21,the relative expression of TLK2 mRNA decreased significantly(P＜0.05).Conclusion:miR-21 is highly expressed in AML patients,which may promote the apoptosis of AML cells by inhibiting the expression of TLK2.

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.
Apoptosis ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Drug Synergism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Real-Time Polymerase Chain Reaction ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Transfection