ObjectiveTo observe the protective effect of Didang Xianxiong decoction on the kidneys of diabetic rats， its regulation on the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， and its influence on podocyte apoptosis and explore the mechanism of Didang Xianxiong decoction in improving diabetic nephropathy. MethodThe diabetic model was established by a single intraperitoneal injection of streptozotocin （STZ） solution of 55 mg·kg^-1. The successfully replicated model rats were randomly divided into the model group， Didang Xianxiong decoction group （8.10 g·kg^-1）， Xiao Xianxiongtang group （4.05 g·kg^-1）， Didangtang group （4.05 g·kg^-1）， and alagebrium （ALT-711） group （3 mg·kg^-1）， with six rats in each group. In addition， six rats were included in the blank group. After continuous administration for eight weeks， hematoxylin-eosin （HE） staining was used to observe the pathological changes in rats' kidney tissue. Masson staining was used to observe the degree of collagen deposition. Periodic acid-Schiff （PAS） staining was used to observe basement membrane lesions， and immunohistochemistry was used to detect the expression of phosphorylation （p）-PI3K and p-Akt proteins in rats' kidney tissue. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） method was used to detect podocyte apoptosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of PI3K and Akt in rats' kidney tissue. Western blot was used to detect the protein expression of PI3K， p-PI3K， Akt， p-Akt， B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， phosphorylation glycogen synthase kinase-3β （p-GSK-3β）， and Caspase-3 in the kidney tissue. ResultCompared with the normal group， the model group had compensatory expansion of glomeruli， proliferation of mesangial cells， a large amount of collagen deposition in the mesangial stroma， thickening of the basement membrane， decreased mRNA expression of PI3K and Akt， and inhibition of PI3K and Akt protein phosphorylation （P<0.01）. It also underwent enhanced apoptotic signaling， decreased expression of anti-apoptotic protein Bcl-2 （P<0.01）， and increased expression of Bax， p-GSK-3β， and Caspase-3 （P<0.01）. Compared with the model group， Didang Xianxiong decoction significantly improved kidney tissue pathology， increased mRNA expression of PI3K and Akt （P<0.01）， significantly up-regulated phosphorylation levels of PI3K and Akt proteins （P<0.01） and Bcl-2 expression （P<0.01）， downregulated the expression of Bax， p-GSK-3β， and Caspase-3 （P<0.01）， and weakened podocyte apoptotic signaling. ConclusionDidang Xianxiong decoction may promote the activation of the PI3K/Akt signaling pathway， inhibit podocyte apoptosis， and thus slow down the progression of diabetic nephropathy.

Objective:To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) .Methods:Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified.Results:① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123 + tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group ( P<0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10 5/ml to 3.2×10 6/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8 + PD-1 + LAG-3 + T cells was 10.90%, and the proportion of propidium iodide (PI) - Annexin Ⅴ + T cells and PI + Annexin Ⅴ + T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group ( P<0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group ( P<0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group ( P<0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123 + tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123 + MV4-11 cells, CD123 + Molm13 cells, and CD123 + THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group ( P<0.05) . Conclusion:In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123 + tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

Objective:To build a systematic and standardized nutrition management plan for patients with chronic kidney disease.Methods:Based on the nutrition care procedure and model, a preliminary draft of a nutrition management plan for chronic kidney disease patients was developed through a literature search, quality evaluation, and group discussions. After two rounds of expert consultation and revision of the preliminary draft of the nutrition management plan, the final plan was formed.Results:A total of 32 experts were invited to complete two rounds of consultation. In two rounds of expert consultation, 32 questionnaires were distributed, and 32 and 31 valid questionnaires were collected, with valid response rates of 100.0% and 96.9%, respectively. The expert authority coefficients were 0.853 and 0.871, respectively. The final nutrition management plan for chronic kidney disease patients included six first-level items of nutrition management personnel: nutrition risk screening, nutrition assessment, nutrition treatment, nutrition monitoring, and nutrition health education, with 23 second-level and 52 third-level items.Conclusions:The constructed nutrition management plan for chronic kidney disease patients is scientific and can provide a reference for nutrition guidance.

Breast cancer was the most commonly diagnosed cancer, with an estimated 2.3 million new cases (11.7%), followed by lung (11.4%) in 2020. Breast cancer ranks first among malignant tumors in the world, seriously threatening women's health. Due to continuously enrichment of treatment methods for breast cancer, patients' prognosis have been greatly improved. The emergence of vaccines is an important treatment method to promote the development of human health. For cancer therapy, preventive vaccines have been popularized for kinds of tumor with specific incentives, such as cervical cancer caused by HPV infection. At present, the causative factors of breast cancer are still unclear, and it is still difficult to develop preventive vaccines against breast cancer. In recent years, a variety of therapeutic vaccines have emerged in the field of breast cancer treatment. When patient completed comprehensive treatment, vaccine is used to stimulate body immune system to recognize tumor cell-specific antigens, thereby reducing the recurrence rate as much as possible. Most of these vaccines are currently aimed at more malignant triple-negative breast cancer and HER2-positive breast cancer. This article will focus on the research progress of several therappeutic vaccines.

【Objective】 To explore the establishment methods of transgenic human umbilical cord mesenchymal stem cells (hUC-MSCs) overexpressing tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) based on the transposons, and attempt to apply it on the nude mice mode with glioma. 【Methods】 PiggyBac transposon system specially designed by us was used to prepare non-targeting and Her2-targeting hUC-MSCs that can stably express TRAIL through puromycin screening. The glioma cells expressing firefly luciferase (U87MG-LUC) were injected into the skull of the immunodeficient mice (BALB/c-nu/nu) with 1×10⁶ cells per mouse. After 7 days of injection, the mice transplanted with U87MG were detected with a small animal living imager to determine the size and location of the tumors in skull. Then we injected the glioma-transplantation nude mouse with two kinds of transgenic hUC-MSCs expressing TRAIL (named as untarget-TRAIL and target-TRAIL, respectively), or the non-transgenic hUC-MSCs (all 1×10⁶ cells per mouse) or PBS (named as WT-MSCs and PBS for negative control) respectively, and then monitored the changes of tumor signals by a small animal living imager every week for 3~4 weeks. 【Results】 After six passages to expand the cells, the both transgenic cell lines can stably express TRAIL gene. Their ratio of green fluorescent protein (GFP) positive cells can reach 93%-97%, and the positive ratio of their MSC-specific surface markers still maintained normal (CD34⁺, CD45⁺, and HLA-DR⁺ all <0.1%, CD90>99%, CD73>88%, and CD105 >60%). The median survival time (d) of U87MG-transplanted nude mice in the groups of untarget-TRAIL, target-TRAIL, WT-MSCs, and PBS was 41 vs 39 vs 24 vs 23(P<0.05). 【Conclusion】 The transgenic hUC-MSCs overexpressing TRAIL gene can significantly prolong the survival time of nude mice with brain glioma.

Objective:To prepare a novel tri-specific T cell engager (19TriTE) targeting CD19 antigen, and to investigate its immunotherapeutic effect on CD19-positive hematological malignancies.Methods:19TriTE was constructed by molecular cloning technology and successfully expressed through the eukaryotic expressing system. The effects of 19TriTE on the proliferation and activation of T cells, as well as the specific cytotoxicity against CD19 positive tumor cell lines were verified.Results:①19TriTE expressing plasmid was constructed and successfully expressed through the eukaryotic expressing system. ②19TriTE can specifically bind to T cells and Nalm6 cells, with equilibrium dissociation constants of 19.21 nmol/L and 11.67 nmol/L, respectively. ③The expression rates of CD69 positive T cells and CD25 positive T cells were 35.4% and 49.8% respectively, when 2 nmol/L 19TriTE were added in the co-culture system, which were significantly higher than those in the control group. ④19TriTE can significantly promote the proliferation of T cells. The absolute count of T cells expanded from the initial one million to 74 million with an 74 fold increase at the concentration of 1 nmol/L on day 12. ⑤19TriTE can significantly mediate T cells killing of CD19 positive target cells in a dose-dependent manner. At the concentration of 10 nmol/L, the target cells lysis reached 50%. ⑥Degranulation experiment verified that 19TriTE can activate T cells in the presence of CD19 positive target cells, and the activation of T cells positively correlated with the dose of 19TriTE. ⑦When 19TriTE fusion protein co-cultured with T cells and target cells overexpression RFP and luciferase genes respectively, 19TriTE can notably mediate T cells killing of CD19 positive target cells through fluorescent microscope or bioluminescence imaging technology.Conclusion:In this study, we successfully constructed and expressed 19TriTE fusion protein and verified that it can effectively activate T cells and promote their proliferation in vitro. At the same time, it can bind to CD19 positive target cells and T cells, as well as enhance T cells anti-leukemia effect in vitro, providing the foundation for further clinical research.

Objective:To investigate the distant metastasis after primary treatment of papillary thyroid cancer (PTC) in children and adolescents.Methods:A retrospective analysis of 180 cases (54 boys and 126 girls, with an age range of 6-18 years) with PTC treated at the Chinese Medical Academy Cancer Hospital and Zhejiang Cancer Hospital from January 1, 2001 to December 31, 2014 was performed. Patients’ clinical and pathological data were collected. The follow-up results were statistically analyzed. The distant metastasis rate during the follow-up period was analyzed by the Kaplan-Meier method. Log-Rank test was used for univariate analysis and Cox regression model was established in multivariate analysis.Results:Twenty-four cases (13.3%) had distant metastases during following-up with a median of 92 months. The Log-Rank test showed that the younger age ≤15 years old (χ 2=11.803, P=0.001), the larger tumor diameter >20 mm (χ 2=5.776, P=0.016), multifocal (χ 2=11.205, P=0.001), bilateral tumor distribution (χ 2=19.804, P=0.001), invaded capsule (χ 2=10.808, P=0.001), and bilateral lymph nodes metastasis (χ 2=6.278, P=0.012) were risk factors for distant metastasis after initial treatment. The Cox regression analysis showed that age ≤15 years (hazard ratio [95% confidence interval]: 4.08[1.504-11.111], P=0.006) and bilateral tumor distribution (hazard ratio [95% confidence interval]: 4.77[1.903-11.966], P=0.001) were independent risk factors for distant metastasis after initial treatment. The risk factors for local recurrence and distant metastasis were similar, but the local recurrence could not be a significant predictor for distant metastasis. It was indicated that distant metastasis rate was lower in patients with total thyroidectomy in multifocal lesions groups (χ 2=5.891, P=0.015). Conclusions:Age, tumor size, invaded capsule, bilateral lymph nodes metastasis, multifocal and bilateral lesions are factors for predicting distant metastasis after primary treatment of PTC in children and adolescents. Total thyroidectomy is recommended for patients with multifocal and bilateral lesions.

Objective To compare the effect of pressurized rapid intravenous drip of Shenfu and micro-pumping of dopamine on hypoperfusion in percutaneous coronary intervention (PCI) of acute inferior wall myocardial infarction.Methods A total of 80 patients with acute inferior wall myocardial infarction treated with emergency PCI in Chest Pain Unit of Yantaishan Hospital of Yantai City from May 2014 to May 2015 were enrolled. Then these patients were divided into observation group (n=40,pressurized rapid intravenous drip of normal saline and Shenfu) and control group (n=40,pressurized intravenous drip of normal saline with the same pressure combined with micro-pumping of Dopamine) according to random number table. Blood pressure and heart rate per five minutes were observed and recorded before intravenous drug,five to forty minutes after intravenous drug. Besides,the incidence rates of malignant arrhythmia,vomitting,TIMI grade three flows after intrqvenous drug and major adverse cardiovascular events after 30 days of a follow-up were also observed and recorded.Results Systolic pressure,diastolic pressure and mean arterial pressure of patients between two groups all met and maintained to the expectation after treatment with significant differences between two groups (P<0.05). The incidence rates of malignant arrhythmia,nausea and vomitting in observation group were lower than those in control group with significant differences (P<0.05). There was no statistically significant difference in the proportion of TIMI grade three flows and the incidence of major adverse cardiovascular events after 30 days of a follow-up (P>0.05).Conclusions When the hypoperfusion appears during PCI of acute inferior wall myocardial infarction,the pressurized rapid intravenous drip of Shenfu can improve hypoperfusion which is better than Dopamine in the effects of correcting hypotension and reducing the occurrence of adverse events.

Objective To establish a novel TRFIA for the measurement of heparanase (HPA) in serum samples,and investigate its clinical application.Methods The micro-pore plate wells were first coated with partially recombinant murine anti-human HPA monoclonal antibody.Biotin-labeled recombinant HPA protein was then used to compete with HPA in serum samples,and the prepared europium (III)-labeled streptavidin (Eu3+-SA) was used as signal readout for establishing the BSA-TRFIA assay.Using this assay,the serum HPA levels in healthy subjects (n=32) and tumor patients (n=54) were measured.The results of BSA-TRFIA were compared with those of ELISA.Two-sample t test (or t' test),and linear correlation analysis were used to analyze the data.Results The sensitivity of BSA-TRFIA for measuring HPA was 0.33 ug/L.The CV values for intra-batch and inter-batch were 5.29% and 7.54%,respectively.The average recovery rate was 105.5%.The standard curve range was 0-1 000 ug/L.The serum HPA level measured by the BSA-TRFIA method in healthy subjects was (2.03_+ 1.47) Iug/L.In tumor patients,the HPA level was significantly higher:(22.13_+7.38) ug/L (t'=19.388,P

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases, including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells, direct cell reprogramming driven by lineage-determining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter, we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover, they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination, but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling, drug screening, and cell therapy of retinal degeneration.
Animals ; Bestrophins ; Cell Differentiation ; Cell Line ; Cell Lineage ; Chloride Channels ; genetics ; metabolism ; Embryonic Stem Cells ; cytology ; metabolism ; Eye Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Mice ; Pigmentation ; Retinal Pigment Epithelium ; cytology ; metabolism ; Transcription Factors ; metabolism