Epidermal growth factor (EGF) is an epithelial cell growth factor that can stimulate intestinal development, repair the damage of epidermal cells as well as reduce the incidence of pathogen infection and diarrhea. In order to produce a recombinant Lactobacillus plantarum (L. plantarum) expressing porcine epidermal growth factor (pEGF), we constructed a recombinant vector stably expressing pEGF in L. plantarum strains. First, L. plantarum strain Lp-1 was isolated from intestinal contents of piglets. Then the functional domain of pEGF, M6 precursor protein signal peptide (SP) and super strong constitutive promoter (SCP) were connected with the backbone plasmid pIAβ8 to construct the recombinant vector that was transformed into Lp-1 by electroporation. Afterwards, pEGF was expressed in Lp-1 and detected by Tricine-SDS-PAGE and ELISA. After orally irrigated early-weaned BALB/c mice with the recombinant L. plantarum every morning and late afternoon for 10 consecutive days, body weight, villous height and crypt depth in the intestine were measured to examine the influence of the recombinant bacteria on the intestinal development of early-weaned mice in vivo. Finally, the results of our experiments demonstrated that pEGF was successfully expressed in Lp-1 and the molecular weight of pEGF was 6 kDa. In addition, the recombinant pEGF can enhanced the daily gain and exerted significance influence (P < 0.05) to the small intestinal morphology of early-weaned BALB/c mice. In conclusion, pEGF could be expressed in L. plantarum and the recombinant pEGF possesses good biological activity.
Animals ; Electrophoresis, Polyacrylamide Gel ; Epidermal Growth Factor ; biosynthesis ; Genetic Vectors ; Intestines ; microbiology ; Lactobacillus plantarum ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; Promoter Regions, Genetic ; Protein Precursors ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis ; Swine

Objective:The main objective of this study is to analyze the development trend of China's private healthcare providers since the issue of Document No. 58 by the General Office of the State Council in the year 2010. It intends to evaluate the effectiveness of the policy on encouragement and guidance to private healthcare. Methods:(1) Using the statistical data collected from national and provincial healthcare yearbook, we made a comparative a-nalysis on seven indicators regarding the development of private healthcare providers, including the number of health-care providers, the number of beds, the number of healthcare professionals, annual outpatient diagnose-and-treat per-son-times, annual inpatient hospitalization person-times, bed utilization rate and average length of stay for two periods of time (i. e. 2006—2010 and 2011—2015). (2) A field study was conducted to six selected provinces. In these provinces, essential information of the related policy was collected, held stakeholder interviews and focus group dis-cussions among hospital management team and medical workers and visits to several typical private providers were made to understand the policy effectiveness and existing problems. The study also tried to find the key factors for a successful private healthcare provider in China. Results:(1) The results show that since the issue of Document No. 58 in the year 2010 , the number of China's private healthcare providers has greatly increased while the scale and service capabilities of private providers still need to be improved. (2) As per the results again, a great difference ex-ists between provinces in terms of private healthcare provider development during 2010 to 2015 . Conclusion:A posi-tive impact of government regulation on the development of private healthcare providers was noticed. However, China's private healthcare providers are still facing many invisible obstacles and challenges. The government needs to put more focus on building a cross-department coordination and supporting regulation system to advance the sustain-able development of private healthcare providers. Moreover, the government needs to cautiously promote the Public-Private-Partnership ( PPP) to improve the effective allocation of resources in the healthcare market and provide essen-tial support to private healthcare providers in solving the problems they meet during their development process.

Objective To compare the curative effect of perindopril and metoprolol in the treatment of diabetic patients with asymptomatic cardiac insufficiency.Methods According to the method of random numbers, 152 diabetic patients with asymptomatic cardiac insufficiency were divided into the observation group and control group,with 76 cases in each group.The observation group was given with perindopril treatment,while control group was given with metoprolol treatment.The cardiac function index,glycosylated hemoglobin,BNP levels,the incidence of cardiac insufficiency and the occurrence of adverse drug reactions before and after treatment were compared.Results There were no significant differences in LVEF,Tei index and E/A between two groups before treatment (all P >0.05 ).Compared with before treatment,the cardiac function indexes of the two groups were significantly improved after treatment(t=5.96,4.83,4.91,10.22,5.68,5.18,all P<0.05),but the observation group were improved more significantly(t=5.64,4.67,5.03,all P<0.05).Before and after treatment,there was no significant difference in the levels of glycosylated hemoglobin(all P>0.05),and the difference between the two groups was not statistically significant(all P>0.05).The BNP levels of the observation group before and after the treatment were (515.43 ± 58.85)pg/mL and (214.43 ±32.42)pg/mL respectively,of which the control group were (513.94 ±59.11)pg/mL and (263.32 ±43.31)pg/mL respectively,and after treatment,the BNP levels of the two groups were significantly decreased(t=23.45,17.68,all P<0.05),of which the observation group decreased more obvious(t=6.97,P<0.05).The cardiac insufficiency incidence of the observation group was 3.95%,which was significantly lower than 14.47% of the control group(χ2 =5.03,P<0.05).The incidence of adverse drug reactions had no obvious differ-ences in both groups(χ2 =0.08,P>0.05).Conclusion Using perindopril in treatment of diabetic patients with asymptomatic heart function can obviously improve cardiac function,reduce the incidence of BNP and cardiac insuffi-ciency.It has the clinical curative effect and high safety.

Objective : To investigate the clinical and molecular epidemiological characteristics of 10 patients with enterovirus (EV)-D68 infections in the respiratory tract in Shaoxing City, Zhejiang Province, so as to provide insights into formulation of control interventions for EV-D68 infections. Methods: Clinical specimens were sampled from patients with acute respiratory tract infections (ARTIs) admitted to sentinel hospitals in Shaoxing City from 2021 to 2022, and EV-D68 was detected using real-time fluorescent PCR assay and sequencing of the VP1 region of enterovirus. The epidemiological and etiological characteristics of EV-D68 infected cases were descriptively analyzed. Results: A total of 3 009 specimens were sampled from patients with ARTIs from 2021 to 2022, and the detection of EV-D68 was 0.33%. Of all EV-D68-infected patients, there were 6 men and 4 women, and 5 cases under 18 years of age, 2 cases at ages of 18 to 60 years and 3 cases at ages of over 60 years. EV-D68 infection predominantly occurred in summer (5 cases detected between May and July) and autumn (5 cases detected between September and October). The main clinical symptoms included fever (10 cases), sore throat (9 cases) and cough (8 cases), and all 10 cases recovered well, with no deaths reported. Sequencing identified D3 subtype in all 10 specimens positive for EV-D68. Conclusions The ARTIs caused by EV-D68 occurred predominantly among children under 18 years of age in Shaoxing City, and was highly prevalent in summer and autumn. D3 was the predominant enterovirus subtype.

OBJECTIVEThe purposes of this study were to investigate effects of chloroqquine (CQ) on inhibiting the decomposition of hydrogen peroxides (HP), and to optimize composition of the tooth-bleaching agent.

METHODSAccording to the principle of the color-changing reaction between horse radish peroxidase and substrate, the tooth-bleaching agent made of HP was divided into four groups with different amounts of CQ. The stability of HP was observed using ELISA for 3 months.

RESULTSWith the prolongation of store time, the absorbance of HP solutions containing different concentrations of CQ declined at different degrees, which showed a dependent relation between absorbance values and concentrations of CQ. Within the range of experiment concentrations of CQ, the higher the concentration of CQ was, the stronger the stability of HP was. And the duration of tooth-bleaching effects in 150.0 mg/ml of CQ was prolonged 4 to 6 times compared to that without CQ.

CONCLUSIONThe data indicate that CQ can inhibit the decomposition of HP. The bleaching effect of the tooth-bleaching agent which is made of HP and proper amount of CQ is satisfactory.

Objective:To study the independent risk factors of tumor recurrence after radiofrequency ablation (RFA) in patients with hepatocellular carcinoma (HCC), and to establish a preoperative prediction score.Methods:A retrospective study was conducted on 168 HCC patients treated with RFA at Zhongshan Hospital affiliated to Sun Yat-sen University from June 2016 to September 2019. The X-tile software was used to determine the optimal cut-off value of preoperative circulating tumor cells (CTC) which was then used to analyze the relationship between different CTCs values with various clinical factors. The Cox regression model was used to analyze independent risk factors of recurrence after RFA, and each independent risk factor was assigned a score of 1 to compose the prediction score. The patients were divided into the low-risk group (0-2 scores), intermediate-risk group (3 scores) and high-risk group (4-5 scores). The Kaplan-Meier method was used to draw cumulative recurrence curves in calculating the cumulative recurrence rates of the 3 different groups.Results:Of 168 patients, there were 151 males and 17 females. Their age (Mean±SD) was 58.33±9.53 years. CTC≥1/3.2 ml was detected in 131 patients (77.98%) (range 0-20/3.2 ml). The X-tile software determined the preoperative CTC cut-off value of HCC patients to be 2/3.2ml which separated a CTC-negative group with 93 patients, and a positive group of 75 patients. On analyses, the relationship between preoperative CTC and various preoperative clinical parameters were related to number of tumor nodules, tumor maximum diameter and alpha-fetoprotein (AFP) levels ( P<0.05). Multivariate analysis showed that CTC positivity[ HR(95% CI): 1.990(1.332-2.974)], AFP>20 ng/ml[ HR(95% CI): 1.659(1.111-2.477)], PIVKA-II>40 mAU/ml[ HR(95% CI): 1.580 (1.022-2.443)], number of tumor nodules ≥2[ HR(95% CI): 1.568 (1.057-2.326)], and tumor diameter>30 mm[ HR (95% CI): 1.544 (1.007-2.369)] were independent risk factors of recurrence ( P<0.05) after RFA in HCC patients. The cumulative recurrence rates of patients at 6 months, 12 months, and 18 months were 14.9%, 35.6%, and 56.4% in the low-risk group, 38.9%, 70.5%, and 85.0% in the intermediate-risk group, and 64.5%, 84.5% and 100% in the high-risk group. The differences were significant ( P<0.05). Conclusion:Preoperative CTC positivity, AFP>20 ng/ml, PIVKA-II>40 mAU/ml, tumor nodules ≥2, and tumor diameter>30 mm were independent risk factors of recurrence after RFA in HCC patients. This preoperative predictive score could be used to guide clinical treatment strategies.

Objective:To explore the application value of detecting circulating tumor cells (CTC) before liver transplantation for predicting the recurrence and survival of hepatocellular carcinoma (HCC).Methods:From October 2015 to October 2019, 62 HCC patients at Affiliated Zhongshan Hospital were collected and analyzed by Cyttel method before liver transplantation. CTC was determined by X-tile software and Kaplan-Meier method for determining the optimal cutoff value of CTC before liver transplantation and the relationship between CTC and clinical factors was analyzed. Univariate and multivariate COX regression analyses were performed for determining the independent risk factors affecting the prognosis. Kaplan Meier method was employed for describing the survival curve of tumor-free survival and overall survival after transplantation.Results:The optimal preoperative critical value of CTC was 3.2 ml. CTC ≥3/3.2 mL was set as CTC positive group while CTC <3/3.2 mL CTC negative group. The positive/negative CTC before transplantation was significantly correlated with preoperative Alpha-fetoprotein(AFP) level, maximal tumor diameter, lymph node metastasis, liver transplantation criteria and degree of differentiation ( P<0.05). Univariate and multivariate COX regression models indicated that the number of preoperative CTC (HR: 1.262, 95%CI: 1.069-1.489, P=0.006) and microvascular invasion (HR: 2.657, 95%CI: 1.120-6.305, P=0.027) were independent risk factors for tumor-free survival after transplantation while microvascular invasion (HR: 3.738, 95%CI: 1.219-11.459, P=0.027) was the sole independent risk factor affecting the overall survival of HCC after transplantation. Statistically significant difference existed between preoperative CTC positive/negative and tumor recurrence or metastasis (no recurrence, intrahepatic recurrence, and distant metastasis)( χ2=7.790, P=0.020). The disease-free survival rates of 1/2/3-year CTC-negative/positive patients were 82.90%, 68.70%, 58.90% and 49.00%, 29.40%, 22.10%; the 1/2/3-year overall survival rates of preoperative CTC-negative/positive patients were 85.50%, 77.10%, 69.79% and 64.90%, 47.20%, 40.50% respectively. The disease-free survival curve of CTC-negative patients was significantly higher than that of CTC-positive counterparts ( P<0.001) and the overall survival curve of CTC-negative patients was significantly higher than that of CTC-positive counterparts ( P<0.005). Conclusions:Preoperative CTC detection has certain application value in evaluating the prognosis of liver cancer after liver transplantation, which has important clinical significance and application prospects.

Objective: To investigate the epidemiological and etiological characteristics of viral diarrhea among children under 5 years of age in Shaoxing City, Zhejiang Province, so as to provide insights into management of viral diarrhea. Methods: The surveillance data on viral diarrhea among children under 5 years of age in Shaoxing City from 2019 to 2022 were collected, including demographics and stool testing results. The epidemiological and etiological characteristics of viral diarrhea were analyzed using a descriptive epidemiological method. Results: A total of 763 diarrheal children under 5 years of age were reported in Shaoxing City from 2019 to 2022, and 236 children were tested positive for virus (30.93%). The detection of virus was 49.01%, 31.61%, 20.43% and 21.89% from 2019 to 2022, which appeared an overall tendency towards a decline (P<0.05). The incidence of viral diarrhea peaked from November to March of the next year and from May to July, and the detection of virus was lower among children with diarrhea living in urban areas (Yuecheng District, Keqiao District and Shangyu District) than among those in suburb areas (Zhuji City, Shengzhou City and Xinchang County) (30.22% vs. 52.00%, P<0.05). There were 206 children tested positive for a single virus, and the detection rates of rotavirus (RV), enteric adenovirus (EAdV), norovirus (NoV), and sapovirus (SaV) were 9.57%, 8.91%, 8.39%, and 0.13%, respectively. There were 25 children with virus co-infections, and the positive rates of EAdV and NoV, RV and EAdV and RV and NoV co-infections were 1.31%, 1.18% and 0.79%, respectively. There were 5 children with triple infections of RV, EAdV and NoV (0.66%). The highest detection of EAdV was seen in April, the highest detection of RV and NoV was seen in January, while SaV was only detected in April. Conclusion The incidence of viral diarrhea among children under 5 years of age peaked in winter and spring in Shaoxing City from 2019 to 2022, and the cases predominantly occurred in urban areas. The detection of virus appeared an overall tendency towards a decline, with high detection of RV, EAdV and NoV.

Objective:To analyze the risk factors of hepatocellular carcinoma microvascular invasion (MVI) and to construct a preoperative prediction clinical scoring system.Methods:A retrospective analysis was made on 113 patients with hepatocellular carcinoma undergoing hepatectomy at Zhongshan Hospital from March 2018 to Jun 2019.Postoperative pathology confirmed 35 cases with microvascular invasion.Results:The multivariate logistic regression model showed that the maximum tumor diameter( OR: 1.028, 95% CI: 1.001-1.005), the smoothness of the capsule edge( OR: 0.208, 95% CI: 0.062-0.699), the positive circulating tumor cells (CTC)( OR: 3.728, 95% CI: 1.029-13.501) and abnormal prothrombin(PIVKA-Ⅱ)( OR: 1.001, 95% CI: 1.000-1.002) were risk factors for MVI. The area, sensitivity and specificity of the clinical score constructed by assigning 1 point to each risk factor were 0.906, 74.29% and 92.31%, respectively. Clinical scores of 0, 1, 2, 3, and 4 predict MVI positive rates of 0 (0/26), 9.09% (3/33), 28.57% (6/21), 77.78% (14/ 18), 85.71% (12/14). Conclusions:Tumor maximum diameter>62 mm, PIVKA-Ⅱ>115 mAU/ml, unsmooth tumor capsule and CTC in peripheral blood are independent high risk factors in patients with MVI.

Objective To investigate the effects of T. gondii soluble tachyzoite antigen (STAgs) on the survival time of mouse heart allograft and the possible mechanism. Methods The STAgs were prepared by pulverizing T. gondii tachyzoite with ultrasound on ice. Cervical heterotopic heart transplantations were done by using Balb/c mice as donors, and C57BL/6 mice as recipients.The recipients were classified randomly into three groups: syngeneic group, acute rejection group and STAgs-treated group. The recipients in acute rejection group and STAgs-treated group were injected subcutaneously with 0. 1 ml PBS and 0. 1 ml (5 μg) STAgs at the 4th day before transplantation respectively, and those in syngeneic group were not subjected to any treatment. The grafts were observed daily by cervical palpation, and the total cessation of cardiac contraction was defined as the endpoint. The heart allografts were harvested at the 7th day after transplantation for pathological examination and immunohistochemical staining for CD4+ T, CD8+ T. Results The recipients in syngeneic group were all alive at the 100th day after transplantation. The average survival time in acute rejection group and STAgs-treated group was (6.7± 0.5) days and (70.8± 3.5) days,respectively (P＜0.05). HE staining showed that the rejection on the 7th day after transplantation in syngeneic group, acute rejection group and STAgs-treated group was fallen into 0 degree, Ⅲ-Ⅳ degree and 0- Ⅰ degree, respectively. Immunohistochemical staining revealed that the CD4+ T and CD8+T were markedly down-regulated in STAgs-treated group as compared with those in acute rejection group. Conclusion T. gondii STAgs can significantly prolong the survival time of mouse heart allograft and inhibit the rejection probably by changing the ratio of TH1/TH2, or inhibiting the effect of dendritic cells by inducing the lipoxin A4.