Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Objective:To explore the application value of part-cut jejunal transection in digestive tract reconstruction of totally laparoscopic total gastrectomy.Methods:The propensity score matching and retrospective cohort study was conducted. The clinicopathological data of 112 patients with gastric cancer who underwent totally laparoscopic total gastrectomy in The Second Affiliated Hospital of Anhui Medical University from June 2018 to September 2022 were collected. There were 81 males and 31 females, aged (70±8)years. Among the 112 patients, 60 patients undergoing diges-tive tract reconstruction by Roux-en-Y anastomosis with part-cut jejunum were set as the part-cut group, and 52 patients undergoing digestive tract reconstruction by traditional Roux-en-Y anasto-mosis were set as the traditional group. Observation indicators: (1) propensity score matching status and comparison of clinical data of patients between the two groups after matching; (2) intraopera-tive and postoperative conditions; (3) follow-up. Comparison of measurement data with normal dis-tribution between groups was conducted using the independent sample t test. Comparison of count data between groups was conducted using the chi-square test or Fisher exact probability. Com-parison of ordinal data was conducted using the nonparametic rank sum test. Propensity score matching was performed using the 1∶1 nearest neighbor matching method, with the caliper value of 0.02. Results:(1) Propensity score matching status and comparison of clinical data of patients between the two groups after matching. Of the 112 patients, 90 patients were successfully matched, with 45 cases in each of the part-cut group and the traditional group. After propensity score matching, the elimination of body mass index, clinical TNM staging confounding bias ensured comparability. (2) Intraoperative and postoperative conditions. After propensity score matching, the total operation time and digestive tract reconstruction time of patients in the part-cut group were (217.0±15.1)minutes and (34.7±1.8)minutes, versus (252.6±21.9)minutes and (52.6±7.4)minutes in the traditional group, respectively, showing significant differences in the above indicators between the two groups ( t=?8.97, ?15.66, P<0.05). (3) Follow-up. After propensity score matching, 90 patients were followed up postoperatively for (47±15)months. During the follow-up, no patient in either group received secondary surgery, and there was no death. There were 3 cases and 10 cases of Roux stasis syndrome in the part-cut group and the traditional group, respectively, showing a significant difference between the two groups ( χ2=4.41, P<0.05). Conclusion:Compared with traditional Roux-en-Y anastomosis, the Roux-en-Y anastomosis with part-cut jejunum in totally laparoscopic total gastrectomy can signifi-cantly shorten the time for digestive tract reconstruction and reduce the incidence of postoperative Roux stasis syndrome.

OBJECTIVE: To investigate the effect of the sequence of intermediate instrumentation and distraction-reduction of the injured vertebra on the surgical efficacy of short-segment percutaneous pedicle screw fixation for thoracolumbar burst fractures with high rate of spinal canal encroachment. METHODS: From January 2016 to January 2022, 38 patients with thoracolumbar burst fractures with high rate of spinal canal encroachment (spinal canal encroachment rate >40%, complete posterior longitudinal ligament, no flipping bone block in the posterior marginal of the vertebra) without spinal cord injury who were were treated with short-segment percutaneous pedicle screw fixation were retrospectively analyzed. During the operation, 18 cases were used distraction-reduction first and then intermediate instrumentation on injured vertebral and sequential distraction-reduction again(the distraction-reduction first group) including 8 females and 10 males with a mean age of 46.5 (38.5, 50.0) years old, and the other 20 cases were used intermediate instrumentation on injured vertebral first and then direct distraction-reduction(the intermediate instrumentation first group) including 10 males and 10 females with a mean age of 46.0 (35.8, 50.8) years. The anterior height ratio of the injured vertebra, local Cobb's angle of the injured vertebrae, the spinal canal encroachment rate, and the improvement rate of spinal canal encroachment were compared and evaluated. RESULTS: All patients were followed up for more than 1 year, and no complications such as spinal cord and root injury, screw loosening and screw rod fracture were found. The anterior height ratio of the injured vertebra, local Cobb' angle of the injured vertebra in the two groups were significantly improved compared with preoperative data(P<0.05), and those at 3 months and 1 year after operation was lost compared with that at the previous time point(P<0.05). Although the spinal canal encroachment rate of the two groups 1 day and 1 year after operation was improved compared with that before operation(P<0.05), the improvement of spinal canal volume in the distraction-reduction first group was significantly better than that in the intermediate instrumentation first group (P<0.01). CONCLUSION In the treatment of patients with thoracolumbar fractures with high rate of spinal canal encroachment, short-segment percutaneous pedicle screw internal fixation with distraction-reduction first and then intermediate instrumentation and sequential distraction-reduction again can more effectively reduce the bony encroachment in the spinal canal and achieve indirect decompression effect better.
Humans ; Female ; Male ; Adult ; Middle Aged ; Spinal Fractures/surgery* ; Thoracic Vertebrae/surgery* ; Lumbar Vertebrae/surgery* ; Fracture Fixation, Internal/instrumentation* ; Pedicle Screws ; Retrospective Studies ; Spinal Canal/surgery*

OBJECTIVE: To elucidate the effect of Huanglian-Renshen-Decoction (HRD) on ameliorating type 2 diabetes mellitus by maintaining islet β -cell identity through regulating paracrine and endocrine glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP-1R) in both islet and intestine. METHODS: The db/db mice were divided into the model (distilled water), low-dose HRD (LHRD, 3 g/kg), high-dose HRD (HHRD, 6 g/kg), and liraglutide (400 µ g/kg) groups using a random number table, 8 mice in each group. The db/m mice were used as the control group (n=8, distilled water). The entire treatment of mice lasted for 6 weeks. Blood insulin, glucose, and GLP-1 levels were quantified using enzyme-linked immunosorbent assay kits. The proliferation and apoptosis factors of islet cells were determined by immunohistochemistry (IHC) and immunofluorescence (IF) staining. Then, GLP-1, GLP-1R, prohormone convertase 1/3 (PC1/3), PC2, v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafA), and pancreatic and duodenal homeobox 1 (PDX1) were detected by Western blot, IHC, IF, and real-time quantitative polymerase chain reaction, respectively. RESULTS: HRD reduced the weight and blood glucose of the db/db mice, and improved insulin sensitivity at the same time (P<0.05 or P<0.01). HRD also promoted mice to secrete more insulin and less glucagon (P<0.05 or P<0.01). Moreover, it also increased the number of islet β cell and decreased islet α cell mass (P<0.01). After HRD treatment, the levels of GLP-1, GLP-1R, PC1/3, PC2, MafA, and PDX1 in the pancreas and intestine significantly increased (P<0.05 or P<0.01). CONCLUSION HRD can maintain the normal function and identity of islet β cell, and the underlying mechanism is related to promoting the paracrine and endocrine activation of GLP-1 in pancreas and intestine.
Animals ; Glucagon-Like Peptide 1/metabolism* ; Diabetes Mellitus, Type 2/metabolism* ; Glucagon-Like Peptide-1 Receptor/metabolism* ; Insulin-Secreting Cells/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Male ; Blood Glucose/metabolism* ; Insulin/blood* ; Mice ; Intestinal Mucosa/pathology* ; Apoptosis/drug effects* ; Cell Proliferation/drug effects* ; Islets of Langerhans/pathology*

Objective:To explore the application value of part-cut jejunal transection in digestive tract reconstruction of totally laparoscopic total gastrectomy.Methods:The propensity score matching and retrospective cohort study was conducted. The clinicopathological data of 112 patients with gastric cancer who underwent totally laparoscopic total gastrectomy in The Second Affiliated Hospital of Anhui Medical University from June 2018 to September 2022 were collected. There were 81 males and 31 females, aged (70±8)years. Among the 112 patients, 60 patients undergoing diges-tive tract reconstruction by Roux-en-Y anastomosis with part-cut jejunum were set as the part-cut group, and 52 patients undergoing digestive tract reconstruction by traditional Roux-en-Y anasto-mosis were set as the traditional group. Observation indicators: (1) propensity score matching status and comparison of clinical data of patients between the two groups after matching; (2) intraopera-tive and postoperative conditions; (3) follow-up. Comparison of measurement data with normal dis-tribution between groups was conducted using the independent sample t test. Comparison of count data between groups was conducted using the chi-square test or Fisher exact probability. Com-parison of ordinal data was conducted using the nonparametic rank sum test. Propensity score matching was performed using the 1∶1 nearest neighbor matching method, with the caliper value of 0.02. Results:(1) Propensity score matching status and comparison of clinical data of patients between the two groups after matching. Of the 112 patients, 90 patients were successfully matched, with 45 cases in each of the part-cut group and the traditional group. After propensity score matching, the elimination of body mass index, clinical TNM staging confounding bias ensured comparability. (2) Intraoperative and postoperative conditions. After propensity score matching, the total operation time and digestive tract reconstruction time of patients in the part-cut group were (217.0±15.1)minutes and (34.7±1.8)minutes, versus (252.6±21.9)minutes and (52.6±7.4)minutes in the traditional group, respectively, showing significant differences in the above indicators between the two groups ( t=?8.97, ?15.66, P<0.05). (3) Follow-up. After propensity score matching, 90 patients were followed up postoperatively for (47±15)months. During the follow-up, no patient in either group received secondary surgery, and there was no death. There were 3 cases and 10 cases of Roux stasis syndrome in the part-cut group and the traditional group, respectively, showing a significant difference between the two groups ( χ2=4.41, P<0.05). Conclusion:Compared with traditional Roux-en-Y anastomosis, the Roux-en-Y anastomosis with part-cut jejunum in totally laparoscopic total gastrectomy can signifi-cantly shorten the time for digestive tract reconstruction and reduce the incidence of postoperative Roux stasis syndrome.

In the past few decades, microbubbles were widely used as ultrasound contrast agents in the field of tumor imaging. With the development of research, ultrasound targeted microbubble destruction technology combined with drug-loaded microbubbles can achieve precise drug release and play a therapeutic role. As a micron-scale carrier, microbubbles are difficult to penetrate the endothelial cell space of tumors, and nano-scale drug delivery system—nanobubbles came into being. The structure of the two is similar, but the difference in size highlights the unique advantages of nanobubbles in drug delivery. Based on the classification principle of shell materials, this review summarized micro/nanobubbles used for ultrasound diagnosis or treatment and discussed the possible development directions, providing references for the subsequent development.

Objective:To construct the quality evaluation scale of specification of management for humanistic caring in outpatients and test its reliability and validity.Methods:Referring to the group standards in Specification of Management for Humanistic Caring in Outpatients released by the China Association for Life Care,as well as relevant guidelines and literature,a pool of items for the quality evaluation scale of specification of management for humanistic caring in outpatients was formed.After expert consultation and expert argumentation,a quality evaluation scale of specification of management for humanistic caring in outpatients was constructed.From January to February 2024,243 hospital managers from 5 hospitals in Zhengzhou were selected as survey subjects to conduct item analysis,and reliability and validity testing on the scale.Results:Two rounds of expert inquiry and two rounds of expert argumentation were conducted,with questionnaire response rates of 92.00%and 100.00%,respectively,and expert authority coefficients of 0.952.In the second round of the expert inquiry scale,the mean importance score of the first-level indicators was 4.80 to 5.00,the full score ratio was 88.00%to 100.00%,the coefficient of variation was 0.04 to 0.17,and Kendall's coefficient of concordance was 0.857(P＜0.001);the mean importance score of the second-level indicators was 4.60 to 5.00,the full score ratio was 80.00%to 100.00%,the coefficient of variation was 0.00 to 0.21,and Kendall's coefficient of concordance was 0.775(P＜0.001);the mean importance score of the third-level indicators was 4.60 to 5.00,the full score ratio was 76.00%to 100.00%,the coefficient of variation was 0.00 to 0.21,and Kendall's coefficient of concordance was 0.830(P＜0.001).Finally,a quality evaluation scale of specification of management for humanistic caring in outpatients was formed,including 5 first-level indicators,25 second-level indicators,and 58 third-level indicators.Exploratory factor analysis produced 5 common factors with a cumulative variance contribution rate of 74.628%.The Pearson correlation coefficients between the five-factor scores ranged from 0.648 to 0.798,and the correlation coefficients between the factor scores and the total score of the scale ranged from 0.784 to 0.938.The scale-level content validity index(S-CVI)of the scale was 0.945,the item-content validity index(I-CVI)was 0.725 to 1.000,the Cronbach's alpha coefficient of the total scale was 0.973,and the retest reliability coefficient was 0.934.Conclusion:The constructed quality evaluation scale of specification of management for humanistic caring in outpatients has good scientific validity and reliability,and can be used as an evaluation tool for specification of management for humanistic caring in outpatients.

Objective To investigate and analyze the distribution of serum cytokeratin 19 fragment antigen 21-1(CYFRA21-1)and squamous cell carcinoma-associated antigen(SCCA)levels in healthy pregnant women during pregnancy and to assess their diagnostic value for cervical cancer in pregnancy.Methods A total of 441 healthy pregnant women and 69 patients with cervical cancer in pregnancy who attended the Obstetrics and Gynecology Hospital of Fudan University from Jan 2021 to May 2024 were selected,and 165 healthy women in the Physical Examination Center of the Obstetrics and Gynecology Hospital of Fudan University were included in the same period as the control group.The healthy pregnant women were divided into 143 in early pregnancy(T1 group),147 in middle pregnancy(T2)and 151 in late pregnancy(T3).Serum CYFRA21-1 and SCCA values were detected and analyzed in all groups.One-way ANOVA,independent samples t-test,Mann-Whitney U-test,Kruskal-Wallis H-test,logistic analysis,and ROC curves were used for comparative analysis.Results The CYFRA21-1 and SCCA values were 1.66(1.19-2.17)ng/mL and 0.8(0.6-1.0)ng/mL in the control group,3.07(2.11-4.14)ng/mL and 0.9(0.7-1.3)ng/mL in the healthy pregnant women group,and were 4.33(2.99-7.60)ng/mL and 1.8(0.9-8.5)ng/mL in the patients with cervical cancer in pregnancy group,respectively.There was a statistically significant difference in the two serum values between every two groups(P<0.05).CYFRA21-1 levels were 3.13(2.46-4.05)ng/mL,1.89(1.50-2.53)ng/mL and 4.19(3.48-5.43)ng/mL in the T1,T2,and T3 groups,respectively;and SCCA levels were 0.9(0.7-1.1)ng/mL,0.7(0.6-1.0)ng/mL and 1.2(0.8-1.7)ng/mL,respectively.The results of T1 and T3 groups were higher than those of the control group(P<0.05);however,there was no statistically significant difference between the results of the T2 group and those of the control group(P>0.05).The areas under the ROC curves for the diagnosis of cervical cancer in pregnancy for CYFRA21-1,SCCA,human epididymis protein 4(HE4),anti-carcinoembryonic antigen(CEA)and joint indicators were 0.684,0.724,0.612,0.791 and 0.913,with sensitivities of 36%,48%,38%,57%and 73%,specificities of 96%,97%,89%,86%and 99%,respectively.The cut-off values of each indicator were 6.05 ng/mL,2.60 ng/mL,51.45 pg/mL and 1.75 ng/mL,respectively.Conclusion Serum CYFRA21-1 and SCCA levels were higher in pregnant women during early and late pregnancy compared with non-pregnant individuals,while they were not statistically different from non-pregnant women during mid-trimester.CYFRA21-1 and SCCA have diagnostic value for patients with cervical cancer during pregnancy.

Objective:To make a quantitative analysis of the policy tools of 261 prefectural-level basic medical insurance risk pooling policy text,analyze the distribution characteristics in dimensions including phases,tools and objectives,so as to provide references for the optimization of China's basic medical insurance risk pooling policy.Methods:Content analysis and quantitative analysis method were performed,a three-dimensional framework of"tools(X-dimension)-objectives(Y-dimension)-phases(Z-dimension)"was constructed,which was used to perform multi-dimensional classification and cross-comparison analysis on policy items.Results:According to the X-dimension,environmental and demand-based policy tools were constantly improving,but partial policy sub-tools are seriously missing.From the Y-dimension,policy objectives covered multiple dimensions but failed to complement each other.From the Z-dimension,the use of policy tools in different stages shows the characteristics of gradual progressive and hierarchical diffusion.policy suggestions are put forward:Optimize the combination of various policy tools,improve the adaptability of policy content and objectives,and form inter-dimensional policy synergy.On the basis of the summarized experience in the application of policy tools,providing references for provincial-level basic medical insurance risk pooling in China.

Objective:To investigate the mechanism of downregulated programmed cell death 10 ( PDCD10) expression mediating glioblastoma multiforme (GBM) resistance to temozolomide (TMZ). Methods:U87, LN229 and T98g cell lines were transfected with PDCD10 small interfering RNA or negative small interfering RNA. TMZ-resistant cell lines were constructed using 300 μmol/L TMZ (transfected T98g cell line) and 150 μmol/L TMZ (transfected U87 and LN229 cell lines), respectively: TMZ-resistant U87 cell line transfected with PDCD10 small interfering RNA (shPDCD10-U87-RG cells), TMZ-resistant U87 cell line transfected with negative small interfering RNA (EV-U87-RG cells), shPDCD10-T98g-RG cells, EV-T98g-RG cells, shPDCD10-LN229-RG cells and EV-LN229-RG cells. Flow cytometry and real-time quantitative polymerase chain reaction (qRT-PCR) were used to detect the transfection efficiency of TMZ-resistant cell lines and PDCD10 expressions; MTT assay and colony formation assay were used to verify the drug-resistant ability of TMZ-resistant cell lines. Bioinformatics analysis was performed to detect the correlations of PDCD10 with key genes ( MSH6 and PMS2) in mismatch repair (MMR) system, and drug resistant mechanism was explored by detecting the cell cycle and neurosphere formation ability of drug-resistant cells. Results:(1) qRT-PCR showed that compared with that in EV-U87-RG cells, the PDCD10 expression in shPDCD10-U87-RG cells was statistically down-regulated by (32.85±1.14)% ( t=2.925, P=0.049); compared with that in EV-T98g-RG cells, the PDCD10 expression in shPDCD10-T98g-RG cells was significantly down-regulated by (57.17±1.81)% ( t=3.179, P=0.043); compared with that in EV-LN229-RG cells, the PDCD10 expression in shPDCD10-LN229-RG cells was significantly down-regulated by (33.68±1.34)% ( t=3.085, P=0.045). (2) MTT assay showed that compared with the EV-U87-RG cells, the shPDCD10-U87-RG cells had significantly increased viability ( P<0.05); compared with the EV-T98g-RG cells, the shPDCD10-T98g-RG cells had significantly increased viability ( P<0.05). Among the same kind of cells, the viability 3 d after wash-out was significantly increased compared with that at 72 h after TMZ treatment ( P<0.05). Colony formation assay showed that cell lines with down-regulated PDCD10 expression had higher tumorigenic ability. (3) Compared with EV-U87-RG cells and EV-T98g-RG cells, cells with down-regulated PDCD10 expression (shPDCD10-U87-RG cells and shPDCD10-T98g-RG cells) escaped from TMZ-induced G2/M arrest, resulting in TMZ resistance. (4) Bioinformatics analysis revealed that the PDCD10 expression was positively correlated with MSH6 and PMS2 expressions ( r=0.262, P<0.001; r=0.327, P<0.001); qRT-PCR indicated that downregulated PDCD10 expression caused decreased MSH6 and PMS2 expressions, which disrupted the MMR system. (5) Compared with that by EV-U87 cells, number of neurospheres formed by shPDCD10-U87 cells was significantly increased ( P<0.05); compared with that by EV-U87-RG cells, number of neurospheres formed by shPDCD10-U87-RG cells was significantly increased ( P<0.05). Conclusion:PDCD10 affects the therapeutic sensitivity of GBM to TMZ by arresting cell cycle, disrupting MMR system, and increasing cell stemness.