Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy / 中国实验血液学杂志

OBJECTIVE@#To provide a research basis for a safe and effective cell therapy for β-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression of β-globin.@*METHODS@#The human β-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB, and the transcription and translation of β-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line. Furthermore, the erythroid differentiation of CD34+ cells which were transduced lentiviral virus carrying human β-globin from normal human umbilical cord blood cells and peripheral blood cells of patients with β-thalassemia major were confirmed by colony formation assay, cell smear assay and flow cytometry. The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test.@*RESULTS@#The human β-globin was expressed stably in the MEL cells, and CD34+ cells from health umbilical cord blood as well as PBMC from patient with β-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells. The β-globin expression and differentiation in CD34+ cells were demonstrated successfully in the NSG mouse for about 35 months after post-transplant.@*CONCLUSION@#Stable β-globin expression through the optimization of HS4 from CD34+ in the third generation lentiviral vector is safe and effective for patients with severe β-thalassemia and other β-globin abnormal diseases.

Clinical study of PD-1 monoclonal antibody combined with chemotherapy in the preoperative neoadjuvant treatment of stage ⅢA non-small cell lung cancer / 中华胸心血管外科杂志

Objective:To study the effectiveness and safety of programmed cell death receptor 1(PD-1) monoclonal antibody combined with chemotherapy in the preoperative neoadjuvant treatment of stage ⅢA non-small cell lung cancer(NSCLC).Methods:A total of 65 patients with stage ⅢA NSCLC who underwent preoperative neoadjuvant treatment in our hospital from January 2019 to October 2020 were selected. According to the preoperative neoadjuvant treatment plan, they were divided into control group(31 cases) and observation group(34 cases). Patients in the control group were treated with albumin-bound paclitaxel and cisplatin for injection, and the patients in the observation group were treated with immunotherapy(carrelizumab/sintilizumab) on the basis of the control group, all underwent 2 cycles of preoperative neoadjuvant treatment. Compared the clinical efficacy of imaging, T lymphocyte subsets, drug side effects, surgical resection rate, major pathological remission(MPR), complete pathological remission(pCR) and postoperative complications of the two groups of patients, and analyzed the factors those affected MPR.Results:The clinical efficacy of PR and ORR of imaging in the observation group was better than that of the control group( P<0.05). The positive rate of CD3 + cells, the positive rate of CD4 + cells, the positive rate of CD8 + cells and the ratio of CD4 + /CD8 + cells in the observation group after treatment were higher than those in the control group( P<0.05). The drug toxicity of the observation group was higher than that of the control group in RCCEP/rash, abnormal thyroid function, and abnormal myocardial enzymes( P<0.05). Compared among the observation group(carrelizumab group/sintilizumab group), the toxicity of carrelizumab group was higher than that of sintilizumab group in RCCEP/skin rash, bone marrow suppression and abnormal myocardial enzymes( P<0.05). The MPR and pCR of the observation group were higher than those of the control group( P<0.05). There was no significant difference in surgical resection rate, surgical methods and postoperative complications between the two groups( P>0.05). The results of univariate analysis showed that ECOG score, pathological type, neoadjuvant treatment plan were related to MPR( P<0.05). The results of binary logistic regression analysis showed that ECOG score and neoadjuvant treatment plan were independent risk factors affecting MPR( P<0.05). Conclusion:PD-1 monoclonal antibody combined with chemotherapy can enable patients to obtain better MPR and pCR, and can improve the immune function of patients. But the side effects caused by immunotherapy drugs are worthy of attention, and the side effects are different between different immune drugs.

Inhibition of Escherichia coli nitroreductase by the constituents in Syzygium aromaticum / 中国天然药物

Gut bacterial nitroreductases play an important role in reduction of various nitroaromatic compounds to the corresponding N-nitroso compounds, hydroxylamines or aromatic amines, most of which are carcinogenic and mutagenic agents. Inhibition of gut nitroreductases has been recognized as an attractive approach for reducing mutagen metabolites in the colon, so as to prevent colon diseases. In this study, the inhibitory effects of 55 herbal medicines against Escherichia coli(E. coli) nitroreductase (EcNfsA) were examined. Compared with other herbal extracts, Syzygium aromaticum extract showed superior inhibitory potency toward EcNfsA mediated nitrofurazone reduction. Then, the inhibitory effects of 22 major constituents in Syzygium aromaticum against EcNfsA were evaluted. Compared with other tested natural compounds, ellagic acid, corilagin, betulinic acid, oleanic acid, ursolic acid, urolithin M5 and isorhamnetin were found with strong to moderate inhibitory effect against EcNfsA, with IC50 values ranging from 0.67 to 28.98 mol·L-1. Furthermore, the inhibition kinetic analysis and docking simulation demonstrated that ellagic acid and betulinic acid potently inhibited EcNfsA (Ki < 2 μmol·L -1) in a competitively inhibitory manner, which created strong interactions with the catalytic triad of EcNfsA. In summary, our findings provide new scientific basis for explaining the anti-mutagenic activity of Syzygium aromaticum, where some newly identified EcNfsA inhibitors can be used for developing novel agents to reduce the toxicity induced by bacterial nitroreductase.

PD-1 inhibitor combined with chemotherapy in preoperative neoadjuvant treatment of stage Ⅲ non-small cell lung cancer: A randomized controlled trial / 中国胸心血管外科临床杂志

@#Objective    To evaluate the efficacy and safety of programmed cell death receptor 1 (PD-1) inhibitor combined with chemotherapy in the preoperative neoadjuvant treatment of stage Ⅲ non-small cell lung cancer (NSCLC). Methods    The clinical data of 68 patients with stage Ⅲ NSCLC who underwent preoperative neoadjuvant treatment in our hospital from June 2019 to October 2020 were analyzed and divided into two groups according to a random number table. There were 34 patients in the control group including 19 males and 15 females with an average age of 59.41±4.77 years. In the observation group, there were 34 patients including 21 males and 13 females with an average age of 61.15±6.24 years. The patients in the control group were treated with albumin-bound paclitaxel and cisplatin for injection, and the patients in the observation group were treated with carrelizumab on the basis of the control group, and both groups received 2 cycles of preoperative neoadjuvant therapy. We compared the clinical efficacy of imaging, T lymphocyte subsets, drug side effects, surgical resection rate, major pathological remission (MPR), complete pathological remission (pCR) and postoperative complications of the two groups of patients, and analyzed the influencing factors for MPR. Results    The objective response rate (ORR) of imaging in the observation group (70.6%) was higher than that in the control group (38.2%, P<0.05). The positive rate of CD3+ cells, the positive rate of CD4+ cells, the positive rate of CD8+ cells and the ratio of CD4+/CD8+ cells in the observation group after treatment were higher than those in the control group (P<0.05). The drug toxicity of the observation group was higher than that of the control group in the reactive cutaneouscapillary endothelial proliferation (RCCEP)/rash, abnormal thyroid function, and abnormal myocardial enzymes (P<0.05). The MPR (66.7%) and pCR (51.9%) of the surgical observation group were higher than those of the surgical control group (MPR: 19.2%, pCR: 7.7%, P<0.05). There was no statistical difference in surgical resection rate and postoperative complications between the two groups (P>0.05). Univariate analysis showed that ECOG score, pathological type, neoadjuvant treatment plan and surgical resection were related to MPR (P<0.05). The results of binary logistic regression analysis showed that Eastern Cooperative Oncology Group (ECOG) score and neoadjuvant treatment plan were independent risk factors for MPR (P<0.05). Conclusion    The clinical efficacy of PD-1 inhibitor combined with chemotherapy in the preoperative neoadjuvant treatment of stage Ⅲ NSCLC patients is definite, and it can significantly improve the patients' MPR, pCR and cellular immune function, but the side effects caused by immunotherapy drugs need to be concerned.

Pancreatic lipase inhibitory constituents from Fructus Psoraleae / 中国天然药物

Pancreatic lipase (PL), a crucial enzyme in the digestive system of mammals, has been proven as a therapeutic target to prevent and treat obesity. The purpose of this study is to evaluate and characterize the PL inhibition activities of the major constituents from Fructus Psoraleae (FP), one of the most frequently used Chinese herbs with lipid-lowering activity. To this end, a total of eleven major constituents isolated from Fructus Psoraleae have been obtained and their inhibition potentials against PL have been assayed by a fluorescence-based assay. Among all tested compounds, isobavachalcone, bavachalcone and corylifol A displayed strong inhibition on PL (IC < 10 μmol·L). Inhibition kinetic analyses demonstrated that isobavachalcone, bavachalcone and corylifol A acted as mixed inhibitors against PL-mediated 4-methylumbelliferyl oleate (4-MUO) hydrolysis, with the K values of 1.61, 3.77 and 10.16 μmol·L, respectively. Furthermore, docking simulations indicated that two chalcones (isobavachalcone and bavachalcone) could interact with the key residues located in the catalytic cavity of PL via hydrogen binding and hydrophobic interactions. Collectively, these finding provided solid evidence to support that Fructus Psoraleae contained bioactive compounds with lipid-lowering effects via targeting PL, and also suggested that the chalcones in Fructus Psoraleae could be used as ideal leading compounds to develop novel PL inhibitors.

Progress on glioblastoma multiforme treatment with chimeric antigen receptor T-cells / 中国肿瘤临床

Glioblastoma multiforme (GBM) is the most malignant form of glioma, and its treatment through traditional surgery combined with chemotherapy and radiotherapy has limited efficacy. Chimericantigen receptor T-cells (CAR-T) are recombinant receptors for antigen, which, in a single molecule, redirect and mediateantigen recognition, T-cell activation, and, in the case of second-generation chimeric antigen receptors (CARs) costimulation (CD28 or 4-BB), augment T-cell functionality and persistence. CARs are the focus of attention in emerging treatment options for GBM. This article mainly introduces the development process of CAR-T therapy and the recent success of adoptive transfer of CAR-T cells. Effective targets of the treatment of GBM with CAR-T according to this research are discussed as well. Some of the most extensively studied targets on GBM, especially interleukin-13 receptor α chain variant 2, epidermal growth factor receptor-Ⅷ(EGFRⅧ), human epidermal growth factor receptor 2 (ErbB2), and ephrinA2 receptor (ErbA2), and the different characteristics of each kind of alloantigen-specific CAR-T cells, are the basis for CAR-T therapy and indicate their different characteristics or utilities and the prospect of further clinical research. The discovery of selective expression of interleukin-13 receptor alpha 2 in glioma cells more than 20 years ago prompted the clinical trial of CAR-T therapy in stage I GBM tumors, and the therapy was proven safe and effective. EGFRⅧ is a neoantigen presenting only in cancer cells and glioblastoma stem cells. Its presence is correlated with poor prognosis, and a phase Ⅰ/Ⅱ trial is ongoing at different institutes. ErbB2-specific CARs were also expressed in human Tcells.Adoptive transfer of EphA2 (or ErbB2)-specific T cells resulted in the regression of glioma xenografts. Thus, target-specific CAR-T immunotherapy may be a promising approach for the treatment of different target-positive GBM. Finally, we summarize the application value and challenge of CAR-T cell therapy in the treatment of GBM.

Construction and identification of the recombinant adenovirus vector carrying a small interfering RNA targeting the peroxisome proliferator-activated receptor-γ / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Steroid-induced osteonecrosis of the femoral head (ONFH) is a common clinical disease, with a high disability rate. At present, efficient prevention and treatment of steroid-induced ONFH is still lacking. The peroxisome proliferator-activated receptor-γ (PPARγ) is recognized as an important pathogenic gene for the development of steroid-induced ONFH. RNA interference (RNAi) is a tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target proteins. Therefore, down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.</p><p><b>METHODS</b>According to the principles of siRNA design, three duplex siRNA sequences (971 - 989, 1253 - 1271 and 1367 - 1385) derived from the PPARγ gene (NM_001082148) were synthesized. These duplexes were annealed, purified and ligated into 1.0-cytomegalovirus (CMV) shuttle vector. The shuttle vector was transfected into HEK293 cells. The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.</p><p><b>RESULTS</b>After the annealing of single-strand DNA oligo encoding short hairpin RNA (shRNA) sequences, products were identified by gel electrophoresis. These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367 were constructed. These sequences of these recombinant vectors were confirmed. We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ. After purification, the virus titer was higher than 10(10) plaque forming unit (PFU)/ml.</p><p><b>CONCLUSION</b>In this study, three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ, including shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367, were successfully constructed and high titers of recombinant adenovirus were obtained.</p>

Osteogenic potential of human calcitonin gene-related peptide alpha gene-modified bone marrow mesenchymal stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most of the basic and clinical studies of osteonecrosis of the femoral head (ONFH) are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including nervous system. Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism, and plays key roles in the growth, repair and reconstruction of bone. Calcitonin gene-related peptide (CGRP), which is secreted by peptidergic nerve, is the main mediator of bone metabolism. It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Additionally, it enhances the osteoblast mass and the rate of osteoblast formation, and reduces the bone resorption by acting on osteoblasts and osteoclasts. Hence, we aimed to construct recombinant retrovirus vector pLNCX(2)-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs (BMSCs/pLNCX(2)-hCGRPα) after virus infection.</p><p><b>METHODS</b>The constructed recombinant retrovirus vector pLNCX(2)-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000. Virus was collected for BMSCs infection. The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. The cell proliferation was determined by methyl thiazoleterazolium (MTT) assay. The osteogenic potential of BMSCs was evaluated by alkaline phosphatase (ALP) activity.</p><p><b>RESULTS</b>Both mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX(2)-hCGRPα cells. These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs (P < 0.05).</p><p><b>CONCLUSION</b>BMSCs/pLNCX(2)-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.</p>

The Construction of Lentiviral-delivery Human Interleukin-10 Expression Vector and the Abirritative Effect of Pain on CCI Rats / 中国生物工程杂志

Objective:To construct the lentiviral-vector encoding human interleukin-10 protein(LV-hIL-10) and to observe the effect of LV-hIL-10 on controlling neuropathic pain via intrathecal administration in CCI rats. Methods:hIL-10 gene fragment was isolated and amplified from pCYIL-10 plasmid by PCR, and was cloned into pWPXL. The recombinant plasmid pWPXL-IL-10,envelope plasmid pMD2.G and packaging plasmid psPAX2 were cotransfected into 293T cells, LV-hIL-10 is prepared by concentrating the collected supernatant .At the same time, the empty plasmid pWPXL-GFP,pMD2.G and psPAX2 were cotransfected into 293T cells, LV-GFP is prepared for contrast.135 sheer breed pathogen-free adult male Sprague-Dawley rats divided into 9 arrays at random: CCI models 4 arrays (C0,C1,C2,C3), sham operatived rats 4 arrays (S0,S1,S2,S3) and a normal contrast array (N), each respectively intrathecal injection LV-hIL-10 (C1,S1)、LV-GFP (C2, S2),isotonic Nachloride (C3,S3) and control (no implanted catheters and no administration, C0,S0), the pain threshold of each array and the expression of mRNA and protein of IL-10 in spinal cord,pallium and hippocampus on different time were observed after intrathecal administration LV-hIL-10 in successful CCI model rats . Results:The hIL-10 gene fragment was obtained from pCYIL-10 plasmid, pWPXL-hIL-10 was recombinated successfully. the cloned gene segment was validated by DNA sequencing .High titer(2?1010)and highly purified LV-hIL-10 particles were obtained by three plasmids were cotransfected into 293T cells. The mechanical allodynia and thermal hyperalgesia were alleviated via intrathecal injection LV-hIL-10 in CCI rats. The overexpression of IL-10 were detected in spinal cord,pallium and hippocampus , especially in the spinal cord .Conclusions:The mechanical allodynia and thermal hyperalgesia can be relieved by intrathecal injection LV-hIL-10 in CCI rats.

Dexamethasone-induced adipogenesis in primary marrow stromal cell cultures: mechanism of steroid-induced osteonecrosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In steroid-induced osteonecrosis, hypertrophy and hyperplasia of marrow fat cells and lipid deposition of osteocytes can be found in the femoral head. However, the precise reason is not clear yet. The aim of this study was to observe the effect of dexamethasone (Dex) on differentiation of marrow stromal cells (MSCs), and to investigate the pathobiological mechanism of steroid-induced osteonecrosis.</p><p><b>METHODS</b>MSCs in cultures were treated with increasing concentrations of Dex (0, 10(-9), 10(-8), 10(-7), and 10(-6) mol/L) continuously for 21 days. The cells, which were exposed to 0 mol/L (control) or 10(-7) mol/L Dex for 4 - 21 days, were then cultured for 21 days without Dex. MSCs were stained with Sudan III. Number of adipocytes was counted under a light microscope. The activity of alkaline phosphatase (ALP) of MSCs treated with 0, 10(-8), 10(-7), and 10(-6) mol/L Dex for 12 days, and that treated with 0 mol/L and 10(-7) mol/L Dex for 8, 10, or 12 days were determined. The levels of triglycerides, osteocalcin and cell proliferation of MSCs treated with 0 mol/L and 10(-7) mol/L Dex were detected. The mRNA expression levels of adipose-specific 422 (aP2) gene and osteogenic gene type I collagen in MSCs treated with 0 mol/L and 10(-7) mol/L Dex for 6 days were analyzed by whole-cell dot-blot hybridization. Statistical analysis was performed using Student's t test and analysis of variance. P values less than 0.05 were considered significant statistically.</p><p><b>RESULTS</b>The number of adipocytes in cultures increased with the duration of MSCs' exposure to Dex and the concentration of Dex. The level of ALP activity in the MSCs decreased with concentration of Dex. In the control group, it was 8.69 times of that in the 10(-7) mol/L Dex group on day 12 (t = 20.51, P < 0.001). The level of triglycerides in 10(-7) mol/L Dex group was 3.40 times of that in the control (t = 11.00, P < 0.001). The levels of cell proliferation and osteocalcin in the control were 1.54 and 2.42 times of that in the 10(-7) mol/L Dex group respectively. As compared to the control, the mRNA expression of adipose-specific 422 (aP2) gene in 10(-7) mol/L Dex group was significantly increased (t = 36.48, P < 0.001), and that of osteogenic gene type I collagen was decreased (t = 42.07, P < 0.001).</p><p><b>CONCLUSIONS</b>Dex can directly induce the differentiation of MSCs into a large number of adipocytes and inhibit their osteogenic differentiation, which provide a novel explanation for the pathologic changes of steroid-induced osteonecrosis.</p>

Clinical research of hyperbaric, isobaric, and hypobaric solutions of bupivacaine in continuous spinal anesthesia / 中南大学学报(医学版)

OBJECTIVE@#To compare the anesthesia properities of hyperbaric bupivacaine with those of isobaric and hypobaric solutions when administered in the supine position undergoing hip surgery or lower limb surgery using continuous spinal anesthesia.@*METHODS@#Sixty patients( ASA I approximately III ) scheduled for hip or lower limb surgery were randomly divided into 3 groups with 20 patients in each group: Group A: 0. 375% hyperbaric bupivacaine solutions; Group B :0.375% isobaric bupivacaine solutions; and Group C: 0. 375% hypobaric bupivacaine solutions. The following variables were measured every 2 minutes during the first 30 minutes after the intrathecal injection : the onset time of sensation block, the highest plane of analgesia, the time to reach complete motor blockade, and the plane of analgesia and the extent of lower extremities' movement (modified bromage score, BMS) at different time after the administration. Meanwhile the changes of hemodynamics were recorded.@*RESULTS@#There was no statistical difference among the basic conditions ( P > 0.05). The onset time of sensation block, and the time to reach complete motor blockade, and the time receiving the highest sharp pain sensory block in Group A were significantly shorter than those in Group B and Group C ( P < 0.01 ). The plane of analgesia obtained in the hyperbaric group was significantly higher than in both the isobaric and the hypobaric groups ( P < 0.01). The mean arterial pressure(MAP) , HR in the hyperbaric group decreased significantly after the intrathecal injection( P < 0.05 ).@*CONCLUSION@#The 0.375% Isobaric bupivacaine used during contiuous spinal anesthesia in the supine position produces a suitable and a more "controllable" anesthesia, but a minimum dosage of 10 approximately 12.5 mg is required to obtain adequate anesthesic conditions with moderate hemodynamic changes and satisfying analgesia effects. Under similar conditions, 0. 375% hyperbaric bupivacaine produces major hemodynamic consequences with high cephalad spread and 0. 375% hypobaric bupivacaine has a too long onset time.

Survey and intervention into rural doctors' knowledge on the spread of HIV via blood collection and supply / 中华医院管理杂志

Objective To find out about rural doctors' knowledge on the basic facts about AIDS and the spread of HIV via illegal blood collection and supply and explore viable intervention methods in AIDS prevention and treatment in rural areas. Methods All the rural doctors in a certain township where the epidemic of HTV being spread via blood collection and supply was pretty serious were selected for the survey and data were collected by combining qualitative and quantitative studies. Results The survey of 33 doctors was effective( 100% ) ; above 95% had a correct understanding of the spread of AIDS through sex and blood as well as its contagiousness; 50% did not possess the knowledge that HIV could be spread to the baby by the mother but could not be spread through saliva, sweat, mosquito bites or sharing of bathtubs or toilets; above 80% knew about the objective of the blood organizers in their villages, the peak time of blood selling by the villagers, and the risks of diseases being spread via blood, and were opposed to blood selling; 27.3% once in a while used disposable syringes, and 15.2% discarded or sold used disposable syringes. Conclusion There are severe lurking perils of iatrogenic cross infection in rural areas. Rural doctors are in dire need of formal training in AIDS prevention and treatment knowledge and could be expected to become the key force in AIDS prevention and treatment in rural areas.