Clinical study of Acupoint Pressing Therapy of Zang and Fu for the children with functional constipation and food retention of heat patterns / 国际中医中药杂志

Objective:To observe the clinical efficacy, safety and recurrence rate of Acupoint Pressing Therapy of Zang and Fu in the treatment of children with functional constipation.Methods:A total of 120 children with functional constipation form the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine from January 2020 to June 2021, were divided into two groups randomly, 80 in observation group and 40 in control group. Both groups were treated with routin therpy. The observation group was treated with Acupoint pressing therapy of Zang and Fu, and the control group was treated with Xiaoerhuashi syrup. Both groups were treated for 2 weeks. Before and after treatment, TCM syndromes were scored, daily defecation and spontaneous defecation response were recorded, and the efficacy was evaluated. The patients were followed up for 4 weeks and the recurrence was recorded.Results:Seventh and 14th day after treatment, the effective rates of TCM syndrome efficacy in the observation group were 87.5% (70/80) and 92.5% (74/80), and 62.5% (25/40) and 80.0% (32/40) in the control group. The scores of main symptoms, secondary syndromes and total scores of TCM syndromes in the observation group were significantly lower than those in the control group ( P<0.01 or P<0.05) at 7th day ( t values were 2.90, 2.77 and 3.93) and 14th day ( t values were 4.24, 5.95 and 6.27) after treatment. The effective rates of the observation group was 83.8% (67/80) and that of the control group was 65.0% (26/40). The difference between the two groups was statistically significant ( χ2=5.38, P=0.020). In the follow-up, the recurrence rate of the observation group was 9.5% (7/74) and that of the control group was 18.8% (6/32). There was no significant difference between two groups ( χ2=1.79, P=0.181). Conclusion:Acupoint Pressing Therapy of Zang and Fu has the advantages of curative effect, low recurrence rate and safety in the treatment of children's functional constipation.

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patients immunological status, and found dramatic changes in the IGH within the patients immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones during 2-3 weeks of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34 and IGHV4-39 in COVID-19 patients were more abundant than that of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

The classification and clinicopathological analysis of hepatocellular adenoma based on immunohisto-chemistry / 中华肝胆外科杂志

Objective The subtypes of hepatocellular adenoma (HCA) were classified by immuno-histochemical study, and the clinicopathological characteristics of each subtype were analyzed. Methods From December 2003 to March 2018, 31 cases with HCA were retrieved from the archive files of the Depart-ment of Pathology, Peking Union Medical College Hospital, including 16 male patients and 15 female patients. The age ranged from 16 to 63 years. Hematoxylin and eosin ( HE) and immunohistochemical staining were performed with HCA samples. The subtypes were classified by immunohistochemical staining, and the clinicopathological characteristics of each subtype were analyzed. Results The HCA patients had no obvious and specific clinical symptoms, and most of them were diagnosed during the routine health checkup. All of the 31 patients were treated with surgery, and the complete resection was achieved in 26 cases. The adenomas were mainly in the right lobe of the liver (51. 6% , 16/31), and the solitary adenoma accounted for 54. 8% (17/31). 8 HCA were classified as H-HCA, 14 as I-HCA, 7 as β-HCA, and 2 as U-HCA by immunohistochemistry. H-HCA was characterized histologically by marked steatosis and lobulated contours, lacking L-FABP staining. I-HCA exhibited inflammatory infiltrate, telangiectasia, thickened arteries, more or less obvious ductular reaction, with the positive L-FABP/SAA/CRP staining. β-HCA all showed fibrous capsules, some of adenomas exhibited pseudoglandular structure and nodules in nodule. A strong homogeneous cytoplasmic overexpression of GS and nuclear β-catenin were observed in all β-HCA cases. The staining of SAA/CRP/GS was lacking in the U-HCA. Conclusions HCA is rare and difficult to diagnose in clinic. Final diagnosis relies on histological features, and immunohistochemical examinations need to be used for subtyping classification. Each of the four subtypes has characteristic pathological features.

The influence of the toll-like receptor-4 signaling pathway on IFN-γ and PDGF-AB expressions in the synthesis and secretion of pulmonary artery smooth muscle cells of COPD rats / 实用医学杂志

Objective To study the function of the toll-like receptor-4 (TLR-4) signaling pathway in the synthesis and secretion of pulmonary artery smooth muscle cells of rats with COPD. Methods The primary pulmonary artery smooth muscle cells (PASMCs) of rats with COPD were digested, separated and purified. Then they were randomly divided into control group, LPS group, TLR4 inhibitor group (TAK242) and LPS + TLR4 inhibitor group. RT-PCR, Western blot were used to detect the expression level of TLR-4 and NF-κB among groups. The levels of IFN-γ and PDGF-AB in supernatant with PASMCs in each group were detected by ELISA. Results LPS increased the expression of TLR-4、 NF-κB and the levels of IFN-γ and PDGF-AB. The expression of TLR4, NF-κB and the levels of IFN-γ and PDGF-AB were significantly reduced after inhibiting the expression of TLR4(P < 0.05). Conclusion TLR-4 signaling pathway involved in the inflammatory response and pulmonary vascular remodeling which increased the synthesis and secretion of IFN-γ and PDGF-AB in PASMCs. It provides a theoretical approach for the early intervention of clinical with COPD.

The dose-effect relationship of folic acid on human umbilical vein endothelial cells in vitro / 天津医药

Objective To investigate the protective effects of folic acid on the oxidative damage that ox-LDL (oxi?dized low-density lipoprotein receptor 1) render to human umbilical vein endothelial cells (HUVEC). Methods HUVECs were injured by ox-LDL (120 mg/L) for 24 h while they were incubated with various concentration of folic acid (0,15, 60, 150, 225, 300, 375 nmol/L). Then HUVECs were cultured in media contains same concentration of folic acid but without ox-LDL for 72 hours. Finally, HUVECs were harvested after 24, 48, 72 and 96 h. The morphological changes were observed us?ing inverted microscope and cell viability were examined by MTT. Results Various concentrations of folic acid (0,15, 50, 100, 200 and 500 nmol/L) has no obvious promotion or inhibition effect in growth of normal HUVEC (P>0.05). However, compared with the ox-FA-def group, 150, 225, 300 and 375 nmol/L of folic acid promoted proliferation of HUVECs with 96 and 120 hours of incubations (P < 0.05). Folic acid of 60, 150, 225, 300 and 375 nmol/L promoted the proliferation of HUVECs with 72 h and 96 hours of incubation (P<0.05). Conclusion High dose folic acid can reduce the ox-LDL oxida?tive damage on HUVEC in a concentration dependent manner.

Transgenic mouse model integrating siRNA targeting the foot and mouth disease virus.

We have constructed 2 small interfering RNAs (siRNAs) specifically targeting homogenous 3D and 2B1 regions of 7 serotypes of the foot and mouth disease virus (FMDV) and tested the ability of siRNAs to inhibit virus replication in baby hamster kidney (BHK-21) cells and suckling mice. In this study, we generated transgenic mouse models integrating short hairpin RNA (shRNA) targeting microinfected FMDV. When examined at the 7th passage in transgenic mice, the target gene was still found by PCR to be integrated in the genome. Compared to the control mice, the transgenic mice showed only slightly abnormal pathology when they were infected with the FMDV serotype Asia 1. The number of viruses in the tissues of the transgenic mouse was very low and in some tissues no virus could be detected by immunohistochemistry.

Development of an enzyme immunoassay for urinary pregnanediol-3-glucuronide in a female giant panda (Ailuropoda melanoleuca).

In order to enable monitoring of the reproductive status of the female giant panda after observation of estrus behavior, we developed an enzyme immunoassay (EIA) system for urinary pregnanediol-3-glucuronide (PdG), a progesterone metabolite, using commercial reagents and examined the changes in the urinary concentration of PdG in a female giant panda that showed pseudopregnancy and suspicious pseudopregnancy in 6 consecutive years. The developed EIA system had good reproducibility (intra- and interassay CVs 6.1% and 16.3%, respectively), good parallelism between the standard curve and the dose response curve of serial diluted samples and positive correlation (r=0.836) with the data for PdG in the same samples measured by gas chromatography. Urinary PdG in the female panda showed two phases of increase. The first elevation was observed immediately after estrus with the levels of PdG below 100 ng/Crmg, while the second phase was characterized by a drastic elevation above 100 ng/Crmg until the level began to decrease at the end of pseudopregnancy or suspicious pseudopregnancy. The length of the second phase had wider range than that of the first phase. In the present study, a new EIA assay system for urinary PdG in the female giant panda was developed, and we found that the length of the second phase is unstable in the pseudopregnant and suspicious pseudopregnant giant panda, in contrast with the unstable length of the first phase caused by delayed implantation in the pregnant giant panda.

Inhibition of foot-and-mouth disease virus replication in vitro and in vivo by small interfering RNA.

By using bioinformatics computer programs, all foot-and-mouth disease virus (FMDV) genome sequences in public-domain databases were analyzed. Based on the results of homology analysis, 2 specific small interfering RNA (siRNA) targeting homogenous 3D and 2B1 regions of 7 serotypes of FMDV were prepared and 2 siRNA-expression vectors, pSi-FMD2 and pSi-FMD3, were constructed. The siRNA-expressing vectors were used to test the ability of siRNAs to inhibit virus replication in baby hamster kidney (BHK-21) cells and suckling mice, a commonly used small animal model. The results demonstrated that transfection of BHK-21 cells with siRNA-expressing plasmids significantly weakened the cytopathic effect (CPE). Moreover, BHK-21 cells transiently transfected with short hairpin RNA (shRNA)-expressing plasmids were specifically resistant to the infection of the FMDV serotypes A, O, and Asia I and this the antiviral effects persisted for almost 48 hours. We measured the viral titers, the 50% tissue culture infective dose (TCID50) in cells transfected with anti-FMDV siRNAs was found to be lower than that of the control cells. Furthermore, subcutaneous injection of siRNA-expressing plasmids in the neck of the suckling mice made them less susceptible to infection with O, and Asia I serotypes of FMDV.

Development and evaluation of a rapid enzyme-immunoassay system for measurement of the urinary concentration of estrone-3-glucuronide in a female giant panda (Ailuropoda melanoleuca).

To detect estrus for reproductive management, and to determine the relationship between urinary estrogen and estrous behavior, in a female giant panda, we developed and evaluated a rapid enzyme immunoassay (EIA) system for urinary Estrone-3-glucuronide (E1G) using commercial reagents. The developed EIA system took only around 3 hours, including all procedures to obtain a result. It indicated good reproducibility (intra-assay CV of 5.16%, interassay CV of 15.4%) and sensitivity (lowest standard concentration was 0.0156 ng/ml) for measurement of the urinary concentrations of E1G in the giant panda. There was a positive correlation (r=0.934) with the data for estrone (E1) in the same samples, as measured by radioimmunoassay (RIA) performed in a commercial laboratory. The changes in the E1G concentrations were almost synchronous with the changes in E1 assayed by RIA in urine collected during 4 consecutive estrous seasons. The dynamics of urinary E1G measured by this system highly correlated with the occurrence of the presenting estrous behavior in the giant panda. The above results indicate that this assay system may be normally, rapidly and practically used for measurement of the urinary concentration of E1G in the giant panda.

Polymeric nanoparticles with therapeutic gene for gene therapy: I. Preparation and in vivo gene transfer study / 生物医学工程学杂志

VEGF nanoparticle (VEGF-NP) was prepared by a multi-emulsification technique using a biodegradable poly-dl-lactic-co-glycolic (PLGA) as matrix material. The nanoparticles were characterized for size, VEGF loading capacity, and in vitro release. VEGF-NP and naked VEGF plasmid were intramuscularly injected into the ischemia site of the rabbit chronic hindlimb ischemia model and the efficiency of VEGF-NP as gene delivery carrier for gene therapy in animal model was evaluated. Gene therapuetic effect was assessed evaluated by RT-PCR, immunohistochemistry and angiography assay. The average size of VEGF-NP was around 300 nm. The encapsulation efficiency of VEGF was above 96%. Loading amount of VEGF in the nanoparticles was about 4%. In vitro, nanoparticles maintained sustained-release of VEGF for two weeks. Two weeks post gene injection the capillary density in VEGF-NP group (81.22 per mm2) was significantly higher than that in control group (29.54 mm2). RT-PCR results showed greatly higher VEGF expression in VEGF-NP group (31.79au * mm) than that in naked VEGF group (9.15 au * mm). As a carrier system for gene therapy in animal model, VEGF-NP is much better than naked DNA plasmid. The results demonstrate great possibility of using NP carrier in human gene therapy.

Generation of recombinant human tissue factor pathway inhibitor in E. coli / 生物医学工程学杂志

Tissue factor pathway inhibitor (TFPI) is one of the major physiological inhibitors of the human blood coagulation cascade and may have great potential in the prevention and therapy of diseases caused by thrombus formation. In this study, recombinant human tissue factor was generated in E. coli containing a recombinant vector constructed by inserting TFPI cDNA into pGEX-2T vector. The generated recombinant TFPI (rTFPI) could be simply purified with glutathione-agarose affinity method and maintained its biological function in terms of inhibition of tissue factor and factor Xa.

Nanoparticle as a new gene transferring vector in specific expression gene.

OBJECTIVE: To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference. METHODS: Nanoparticle-DNA complex was prepared with Poly-dl-lactic-co-glycolic acid (PLGA) bearing anti-sense monocyte chemotactic protein-1 (A-MCP-1), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 microg), 6 with A-MCP-1 (200 microg) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed. RESULTS: The package efficiency of the nanoparticle-DNA complex was 0.9%, release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300 nm. SMC genomic DNA PCR showed that A-MCP-1 gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-1 mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced. CONCLUSION: Nanoparticle can act as a vector to transfect specific gene.