Mechanism of Glycyrrhizae Radix et Rhizoma Alleviating Tripterygium wilfordii Polyglycoside Tablets-induced Liver Injury / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of cytochrome P4502D6 （CYP2D6） and cytochrome P4503A4 （CYP3A4）， key enzymes of drug metabolism in liver， on acute liver injury in water extract of Glycyrrhizae Radix et Rhizoma （WEOGRR）. MethodHealthy male Kunming mice were divided into normal group， model group， WEOGRR low-， medium- and high-dose groups （5， 10， 15 g·kg-1·d-1） and positive drug group （diammonium glycyrrhizinate， 75 mg·kg-1·d-1）， with 10 in each group. One week after preventive administration， acute liver injury model was induced by single intragastric administration of 270 mg·kg-1 Tripterygium Glycosides tablets， and samples were collected after 18 h. The pathological changes of liver were observed by hematoxylin-eosin （HE） staining. Serum liver function indexes including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyl transpeptadase （γ-GT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） as well as the levels of oxidative stress indexes including malondialdehyde （MDA） and superoxide dismutase （SOD） in hepatocytes were determined by biochemical method. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to detect the mRNA and protein expression levels of CYP2D6 and CYP3A4， respectively. ResultCompared with normal group， model group had significant hepatocyte swelling and inflammatory cell infiltration （P<0.01）， increased AST， ALT， γ-GT， ALP and TBIL （P<0.05）， elevated MDA and decreased SOD （P<0.01） as well as down-regulated mRNA and protein expression levels of CYP2D6 and CYP3A4 （P<0.05）. Compared with the model group， the normal group had intact liver structure without obvious abnormality， and the WEOGRR groups and positive drug group presented alleviated hepatocyte swelling and inflammatory cell infiltration （P<0.01）， reduced AST， ALT， γ-GT， ALP and TBIL （P<0.01）， lowered MDA and increased SOD （P<0.01） as well as up-regulated expression levels of CYP2D6 and CYP3A4 （P<0.01）. ConclusionThe protective effect of WEOGRR on acute liver injury induced by Tripterygium glycosides tablets may be related to reducing the contents of AST， ALT， γ-GT， ALP and TBIL in serum， inhibiting MDA and increasing the activity of SOD in liver cells， and enhancing the activities of CYP2D6 and CYP3A4， thus accelerating the metabolism of toxic substances.

Single-cell transcriptome analysis reveals the regulatory effects of artesunate on splenic immune cells in polymicrobial sepsis / 药物分析学报

Sepsis is characterized by a severe and life-threatening host immune response to polymicrobial infection accompanied by organ dysfunction.Studies on the therapeutic effect and mechanism of immunomod-ulatory drugs on the sepsis-induced hyperinflammatory or immunosuppression states of various im-mune cells remain limited.This study aimed to investigate the protective effects and underlying mechanism of artesunate(ART)on the splenic microenvironment of cecal ligation and puncture-induced sepsis model mice using single-cell RNA sequencing(scRNA-seq)and experimental validations.The scRNA-seq analysis revealed that ART inhibited the activation of pro-inflammatory macrophages recruited during sepsis.ART could restore neutrophils'chemotaxis and immune function in the septic spleen.It inhibited the activation of T regulatory cells but promoted the cytotoxic function of natural killer cells during sepsis.ART also promoted the differentiation and activity of splenic B cells in mice with sepsis.These results indicated that ART could alleviate the inflammatory and/or immunosuppressive states of various immune cells involved in sepsis to balance the immune homeostasis within the host.Overall,this study provided a comprehensive investigation of the regulatory effect of ART on the splenic microenvironment in sepsis,thus contributing to the application of ART as adjunctive therapy for the clinical treatment of sepsis.

A single-cell landscape of triptolide-associated testicular toxicity in mice / 药物分析学报

Triptolide is a key active component of the widely used traditional Chinese herb medicine Tripterygium wilfordii Hook.F.Although triptolide exerts multiple biological activities and shows promising efficacy in treating inflammatory-related diseases,its well-known safety issues,especially reproductive toxicity has aroused concerns.However,a comprehensive dissection of triptolide-associated testicular toxicity at single cell resolution is still lacking.Here,we observed testicular toxicity after 14 days of triptolide exposure,and then constructed a single-cell transcriptome map of 59,127 cells in mouse testes upon triptolide-treatment.We identified triptolide-associated shared and cell-type specific differentially expressed genes,enriched pathways,and ligand-receptor pairs in different cell types of mouse testes.In addition to the loss of germ cells,our results revealed increased macrophages and the inflammatory response in triptolide-treated mouse testes,suggesting a critical role of inflammation in triptolide-induced testicular injury.We also found increased reactive oxygen species(ROS)signaling and down-regulated pathways associated with spermatid development in somatic cells,especially Leydig and Sertoli cells,in triptolide-treated mice,indicating that dysregulation of these signaling pathways may contribute to triptolide-induced testicular toxicity.Overall,our high-resolution single-cell landscape offers comprehensive information regarding triptolide-associated gene expression profiles in major cell types of mouse testes at single cell resolution,providing an invaluable resource for understanding the underlying mechanism of triptolide-associated testicular injury and additional discoveries of therapeutic targets of triptolide-induced male reproductive toxicity.

Single-cell transcriptome analysis uncovers underlying mechanisms of acute liver injury induced by tripterygium glycosides tablet in mice / 药物分析学报

Tripterygium glycosides tablet(TGT),the classical commercial drug of Tripterygium wilfordii Hook.F.has been effectively used in the treatment of rheumatoid arthritis,nephrotic syndrome,leprosy,Behcet's syndrome,leprosy reaction and autoimmune hepatitis.However,due to its narrow and limited treatment window,TGT-induced organ toxicity(among which liver injury accounts for about 40％of clinical reports)has gained increasing attention.The present study aimed to clarify the cellular and molecular events underlying TGT-induced acute liver injury using single-cell RNA sequencing(scRNA-seq)technology.The TGT-induced acute liver injury mouse model was constructed through short-term TGT exposure and further verified by hematoxylin-eosin staining and liver function-related serum indicators,including alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase and total bilirubin.Using the mouse model,we identified 15 specific subtypes of cells in the liver tissue,including endothelial cells,hepatocytes,cholangiocytes,and hepatic stellate cells.Further analysis indicated that TGT caused a significant inflammatory response in liver endothelial cells at different spatial locations;led to marked inflammatory response,apoptosis and fatty acid metabolism dysfunction in hepatocytes;activated he-patic stellate cells;brought about the activation,inflammation,and phagocytosis of liver capsular macrophages cells;resulted in immune dysfunction of liver lymphocytes;disturbed the intercellular crosstalk in liver microenvironment by regulating various signaling pathways.Thus,these findings elaborate the mechanism underlying TGT-induced acute liver injury,provide new insights into the safe and rational applications in the clinic,and complement the identification of new biomarkers and ther-apeutic targets for liver protection.

Clinicopathologic and molecular genetic featuresof metastatic follicular thyroid carcinoma:analyses of 22 cases / 临床与实验病理学杂志

ABSTARCT Purpose To investigate the clinicopathologic characteristics and genetic mutations of metastatic follicular thy-roid carcinoma(FTC).Methods A total of 22 cases of meta-static FTC were collected,including previous medical history,imaging,treatments and outcomes,and next-generation sequen-cing study and Sanger sequencing were performed in 12 cases.Results There were 16 women and 6 men.Sixteen cases were older than 50 years.Seven cases presented with metastases as the first symptom.Fourteen cases developed metastases 3 to 12 years after thyroid surgery.Sixteen cases developed bone metas-tasis,10 cases had lung metastasis,and 3 cases had brain me-tastasis.Those patients with multiple bone metastases progressed during the follow-up period.The common gene mutations in me-tastases were NRAS p.Q61R(6 cases),HRAS p.Q61R(2 ca-ses)and KRAS p.Q61R(1 case),followed by TERT promoter mutation(8 cases).Other mutated genes included KEL,BRCA1/2,ALK,ROS1,ErbB4,etc.Conclusion FTC has a high misdiagnosis rate.Those diagnosed with FTC should under-go regular systemic examinations to detect potential metastasis,especially in bone,lung,and brain.Further research on the sig-nificance of NRAS and other molecular indicators in FTC metas-tasis will help to better predict its biological behaviors.

Celastrol targeting Nedd4 reduces Nrf2-mediated oxidative stress in astrocytes after ischemic stroke / 药物分析学报

Stroke is the second leading cause of death worldwide,and oxidative stress plays a crucial role.Celastrol exhibits strong antioxidant properties in several diseases;however,whether it can affect oxidation in cerebral ischemic-reperfusion injury(CIRI)remains unclear.This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms.Here,we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2(Nrf2).Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry,we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4(Nedd4)and then released Nrf2 from Nedd4 in astrocytes.Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI,which was significantly blocked by celastrol.Furthermore,by inhibiting oxidative stress and astrocyte activation,celastrol effectively rescued neurons from axon damage and apoptosis.Our study uncovered Nedd4 as a direct target of celastrol,and that celastrol exerts an antioxidative effect on as-trocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.

Hepatic COX1 loss leads to impaired autophagic flux and exacerbates nonalcoholic steatohepatitis

The mechanisms underlying autophagic defects in nonalcoholic steatohepatitis (NASH) remain largely unknown. We aimed to elucidate the roles of hepatic cyclooxygenase 1 (COX1) in autophagy and the pathogenesis of diet-induced steatohepatitis in mice. Human nonalcoholic fatty liver disease (NAFLD) liver samples were used to examine the protein expression of COX1 and the level of autophagy. Cox1Δhepa mice and their wildtype littermates were generated and fed with 3 different NASH models. We found that hepatic COX1 expression was increased in patients with NASH and diet-induced NASH mice models accompanied by impaired autophagy. COX1 was required for basal autophagy in hepatocytes and liver specific COX1 deletion exacerbated steatohepatitis by inhibiting autophagy. Mechanistically, COX1 directly interacted with WD repeat domain, phosphoinositide interacting 2 (WIPI2), which was crucial for autophagosome maturation. Adeno-associated virus (AAV)-mediated rescue of WIPI2 reversed the impaired autophagic flux and improved NASH phenotypes in Cox1Δhepa mice, indicating that COX1 deletion-mediated steatohepatitis was partially dependent on WIPI2-mediated autophagy. In conclusion, we demonstrated a novel role of COX1 in hepatic autophagy that protected against NASH by interacting with WIPI2. Targeting the COX1-WIPI2 axis may be a novel therapeutic strategy for NASH.

A highly efficient protein corona-based proteomic analysis strategy for the discovery of pharmacodynamic biomarkers / 药物分析学报

The composition of serum is extremely complex,which complicates the discovery of new pharmaco-dynamic biomarkers via serum proteome for disease prediction and diagnosis.Recently,nanoparticles have been reported to efficiently reduce the proportion of high-abundance proteins and enrich low-abundance proteins in serum.Here,we synthesized a silica-coated iron oxide nanoparticle and devel-oped a highly efficient and reproducible protein corona(PC)-based proteomic analysis strategy to improve the range of serum proteomic analysis.We identified 1,070 proteins with a median coefficient of variation of 12.56％using PC-based proteomic analysis,which was twice the number of proteins iden-tified by direct digestion.There were also more biological processes enriched with these proteins.We applied this strategy to identify more pharmacodynamic biomarkers on collagen-induced arthritis(CIA)rat model treated with methotrexate(MTX).The bioinformatic results indicated that 485 differentially expressed proteins(DEPs)were found in CIA rats,of which 323 DEPs recovered to near normal levels after treatment with MTX.This strategy can not only help enhance our understanding of the mechanisms of disease and drug action through serum proteomics studies,but also provide more pharmacodynamic biomarkers for disease prediction,diagnosis,and treatment.

Discovery and repurposing of artemisinin / 医学前沿

Malaria is an ancient infectious disease that threatens millions of lives globally even today. The discovery of artemisinin, inspired by traditional Chinese medicine (TCM), has brought in a paradigm shift and been recognized as the "best hope for the treatment of malaria" by World Health Organization. With its high potency and low toxicity, the wide use of artemisinin effectively treats the otherwise drug-resistant parasites and helps many countries, including China, to eventually eradicate malaria. Here, we will first review the initial discovery of artemisinin, an extraordinary journey that was in stark contrast with many drugs in western medicine. We will then discuss how artemisinin and its derivatives could be repurposed to treat cancer, inflammation, immunoregulation-related diseases, and COVID-19. Finally, we will discuss the implications of the "artemisinin story" and how that can better guide the development of TCM today. We believe that artemisinin is just a starting point and TCM will play an even bigger role in healthcare in the 21st century.

Impairment of the autophagy-lysosomal pathway in Alzheimer's diseases: Pathogenic mechanisms and therapeutic potential

Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by memory loss and cognitive dysfunction. The accumulation of misfolded protein aggregates including amyloid beta (Aβ) peptides and microtubule associated protein tau (MAPT/tau) in neuronal cells are hallmarks of AD. So far, the exact underlying mechanisms for the aetiologies of AD have not been fully understood and the effective treatment for AD is limited. Autophagy is an evolutionarily conserved cellular catabolic process by which damaged cellular organelles and protein aggregates are degraded via lysosomes. Recently, there is accumulating evidence linking the impairment of the autophagy-lysosomal pathway with AD pathogenesis. Interestingly, the enhancement of autophagy to remove protein aggregates has been proposed as a promising therapeutic strategy for AD. Here, we first summarize the recent genetic, pathological and experimental studies regarding the impairment of the autophagy-lysosomal pathway in AD. We then describe the interplay between the autophagy-lysosomal pathway and two pathological proteins, Aβ and MAPT/tau, in AD. Finally, we discuss potential therapeutic strategies and small molecules that target the autophagy-lysosomal pathway for AD treatment both in animal models and in clinical trials. Overall, this article highlights the pivotal functions of the autophagy-lysosomal pathway in AD pathogenesis and potential druggable targets in the autophagy-lysosomal pathway for AD treatment.

Celastrol induces ferroptosis in activated HSCs to ameliorate hepatic fibrosis via targeting peroxiredoxins and HO-1

Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron- and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species (ROS) and inducing ferroptosis in activated hepatic stellate cells (HSCs). By using activity-based protein profiling (ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay (CETSA), we show that celastrol directly binds to peroxiredoxins (PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6, through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1 (HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2, PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.

Synergistic effects of autophagy/mitophagy inhibitors and magnolol promote apoptosis and antitumor efficacy

Mitochondria as a signaling platform play crucial roles in deciding cell fate. Many classic anticancer agents are known to trigger cell death through induction of mitochondrial damage. Mitophagy, one selective autophagy, is the key mitochondrial quality control that effectively removes damaged mitochondria. However, the precise roles of mitophagy in tumorigenesis and anticancer agent treatment remain largely unclear. Here, we examined the functional implication of mitophagy in the anticancer properties of magnolol, a natural product isolated from herbal

Traditional Chinese medicine in COVID-19

COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread across the globe, posing an enormous threat to public health and safety. Traditional Chinese medicine (TCM), in combination with Western medicine (WM), has made important and lasting contributions in the battle against COVID-19. In this review, updated clinical effects and potential mechanisms of TCM, presented in newly recognized three distinct phases of the disease, are summarized and discussed. By integrating the available clinical and preclinical evidence, the efficacies and underlying mechanisms of TCM on COVID-19, including the highly recommended three Chinese patent medicines and three Chinese medicine formulas, are described in a panorama. We hope that this comprehensive review not only provides a reference for health care professionals and the public to recognize the significant contributions of TCM for COVID-19, but also serves as an evidence-based in-depth summary and analysis to facilitate understanding the true scientific value of TCM.

Pathological significance of NY-ESO-1 expression in the diagnosis of myxoid liposarcoma / 中华病理学杂志

Objective@#To detect the expression of New York esophageal squamous cell carcinoma antigen 1 (NY-ESO-1) in common types of mesenchymal myxoid tumors, and to investigate its significance in the diagnosis and differential diagnosis of myxoid liposarcoma.@*Methods@#A total of 43 formalin-fixed paraffin-embedded samples of mesenchymal myxoid tumors from the Affiliated Hospital of Qingdao University and Qingdao Municipal Hospital ranging between 2010 and 2017 were selected. NY-ESO-1 expression was detected by immunohistochemical staining. DDIT3 gene status was detected by fluorescence in situ hybridization (FISH). NY-ESO-1 mRNA was detected by reverse transcription-PCR (RT-PCR).@*Results@#Histopathology and FISH results confirmed that there were 11 cases of myxoid liposarcoma and 32 other types (including 7 cases of well-differentiated liposarcoma, 1 dedifferentiated liposarcoma, 3 lipomas, 2 lipoblastomas and 19 non-adipocytic tumors). Immunohistochemical staining showed that the positive expression propotion of NY-ESO-1 in myxoid liposarcoma was 11/11, and the positive location was the cytoplasm and nucleus of lipoblast cells. The expression intensity is higher in regions with round cell differentiation. Among the 32 cases of other mesenchymal myxoid tumors, only one well-differentiated liposarcoma showed positive immunoreactivity for NY-ESO-1. RT-PCR confirmed that 7 cases of myxoid liposarcoma (7/11) and one well-differentiated liposarcoma (1/7) had NY-ESO-1 mRNA expression.@*Conclusions@#NY-ESO-1 is positively expressed in myxoid liposarcoma. It can be served as a useful marker for the diagnosis and differential diagnosis of myxoid liposarcoma.

Clinical application of convolutional neural network in pathological diagnosis of metastatic lymph nodes of gastric cancer / 中华外科杂志

Objective@#To examine the value and clinical application of convolutional neural network in pathological diagnosis of metastatic lymph nodes of gastric cancer.@*Methods@#Totally 124 patients with advanced gastric cancer who underwent radical gastrectomy plus D2 lymphadenectomy at Affiliated Hospital of Qingdao University from July 2016 to December 2018 were selected in the study. According to the chronological order, the first 80 cases were served as learning group. The remaining 44 cases were served as verification group. There were 45 males and 35 females in the study group, with average age of 57.6 years. There were 29 males and 15 females in the validation group, with average age of 9.2 years. The pre-training convolutional neural network architecture Resnet50 was trained and fine-tuned by 21 352 patches with cancer areas and 14 997 patches without cancer areas in the training group. A total of 78 whole-slide image served as a test dataset including positive (n=38) and negative (n=40) lymph nodes. The convolutional neural network computer-aided detection (CNN-CAD) system was used to analyze the ability of convolutional neural network system to screen metastatic lymph nodes at the level of slice by setting threshold, and evaluate the system′s classification accuracy by calculating its sensitivity, specificity, positive predictive value, negative predictive value and area under the receiver operating characteristic curve (AUC).@*Results@#The classification accuracy of CNN-CAD system at slice level was 100%.The AUC for the CNN-CAD system was 0.89. The sensitivity was 0.778, specificity was 0.995, overall accuracy was 0.989. Positive and negative predictive values were 0.822 and 0.994, respectively. The CNN-CAD system achieved the same classification results as pathologists.@*Conclusions@#The CNN-CAD system has been constructed to distinguished benign and malignant lymph node slides with high accuracy and specificity. It could achieve the similar classification results as pathologists.

Clinical research of low-dose rituximab therapy and sequential maintenance for patients with refractory idiopathic thrombocytopenic purpura / 中国医师进修杂志

Objective To investigate the efficacy and safety of low-dose rituximab therapy and sequential maintenance for patients with refractory idiopathic thrombocytopenic purpura. Methods Thirty-three patients with refractory idiopathic thrombocytopenic purpura received intravenous rituximab at the dose of 100 mg once a week for 4 consecutive weeks. Complete blood cell count and serum concentrations of immunoglobulin (IgG,IgM and IgA) were monitored regularly. The numbers of CD3+ and CD19+ CD20+ lymphocyte cells were assayed by flow cytometry before and after therapy. Twenty-five patients with responses(complete response and response) were divided into maintained group (12 patients) and control group (13 patients) by random digits table method. The patients in maintained group were treated with rituximab 100 mg every 6 months. The efficacy of maintenance therapy was evaluated through long-term follow-up. Results The complete response(CR) rate, response (R) rate and no response(NR) rate were 48.48%(16/33), 27.27%(9/33) and 24.24% (8/33), respectively. As a result, total effective rate was 75.76% (25/33). There were no significant changes of peripheral blood white blood cell count,hemoglobin,serum immunoglobulin and CD3+lymphocyte counts before and after treatment (P>0.05). However, CD19+ CD20+ cells were almost depleted in patients treated with rituximab: (3.71±2.64)×106/L vs. (279.33±92.78)×106/L, P<0.01. Five patients suffered from allergic response, and 1 patient developed pneumonia and respiratory failure. The relapse rates of maintained group and control group were 1/12 and 4/13, respectively. Conclusions Treatment with low-dose rituximab may be an effective and safe approach in patients with idiopathic thrombocytopenic purpura. Relapse rates can be decreased through maintenance therapy with refractory low-dose rituximab. However, the optimal therapeutic schedule need further investigation.

Clinical study on 20 cases of acquired thrombotic thrombocytopenic purpura caused by different inducements / 重庆医学

Objective To enhance the recognition of thrombotic thrombocytopenic purpura(TTP) by analyzing the clinical data of acquired TTP .Methods The clinical features ,laboratory detection ,treatment strategy and outcome of 20 patients with ac‐quired TTP were retrospectively analyzed .Results Among 20 patients with acquired TTP ,16 cases had the inducing factors ,inclu‐ding abnormal autoimmune ,systemic lupus erythematosus(SLE) ,antiphospholipid antibody syndrome(APS) ,viral infection ,bacte‐rial infection ,medication ,lymphoma and allogenic hematopoietic stem cell transplantation .When the patients were diagnosed firstly , only 5 cases(25% ) had typical pentalogy of TTP ,18 cases(90% ) were complicated with thrombocytopenia ,14 cases(70% ) were complicated with microangiopathic hemolytic anemia ,17 cases (85% ) with neurologic abnormalities ,15 cases (75% ) fever and 8 ca‐ses(40% ) with renal insufficiency .Sixteen cases(80% ) had high percentage of reticulocytes ,the schistocytes were detected in the peripheral blood smears of 18 cases ,among them microspherocytes were simultaneously detected in 3 cases .Twenty cases had high level of lactate dehydrogenase .All the cases received the therapy of plasmatherapy (plasma exchange and/or plasma infusion) as the principal thing ,among them 9 cases received the glucocorticoid therapy ,2 cases received the cytoxan therapy ,2 cases received the human immune globulin therapy and 4 cases received the low‐dose rituximab therapy (100 mg ,once per week ,for consecutive 4 times) .Eleven cases (55% ) reached complete remission(CR) ,3(15% ) cases got partial remission(PR) and 4(15% ) cases died . The total effective rate was 70% .Three cases(21% ) out of 14 effective cases relapsed .Conclusion The diagnosis of TTP is based on the comprehensive analysis of clinical data .Early diagnosis and early treatment of plasmatherapy as the main means can improve the prognosis of patients with TTP .

Imatinib resistance of K562 cells co-cultured with bone marrow stromal cells from patients with chronic myeloid leukemia / 中国组织工程研究

BACKGROUND:Imatinib resistance is a key issue in treatment of chronic myeloid leukemia. It is confirmed that the leukemia cels can obtain drug resistance phenotype mediated by adhesion to the bone marrow stromal cels (BMSCs). But, the role of BMSCs in imatinib resistance is unclear because chronic myeloid leukemia is deficient in adhesion function. OBJECTIVE: To in vitro simulate the bone marrow microenvironment of patients with chronic myeloid leukemia and to explore its influences on imatinib sensitivity as wel as possible mechanisms. METHODS:BMSCs isolated from patients with chronic myeloid leukemia but not undergoing treatment were co-cultured with K562 cels to construct the BMSCs-K562 cel co-culture model in chronic myeloid leukemia patients, then exposed to 0.2-3.2 μmol/L imatinib for 48 hours, and the proliferation inhibition rate of K562 cels was studied by MTT assay. The apoptosis rate of K562 cels and the expression of the CXCR4 in K562 cels exposed to 0.5 μmol/L imatinib for 72 hours were detected by flow cytometry. The K562 cels were exposed to 0.5 μmol/L imatinib for 4 hours and labeled by Calcein-AM fluorescent labeling sytem, and then, the adhesion rate of the K562 cels was calculated based on fluorescence intensity. RESULTS AND CONCLUSION: The suppressing effect of imatinib (0.2-3.2μmol/L) on the proliferation of K562 cels was weakened significantly by co-culture with the bone marrow stromal cels from patients with chronic myeloid leukemia. The apoptosis rate of K562 cels exposed to 0.5 μmol/L imatinib for 72 hours in co-culture group was significantly lower than that in the suspension culture group (P=0.020). The positive rates of CXCR4 in the co-culture group and suspension culture groups were both increased after exposure to 0.5 μmol/L imatinib for 72 hours (P=0.001). The adhesion rate of the K562 cels to the BMSCs was elevated from (32.18±6.17)% to (68.97±11.08)% when the K562 cels were exposed to 0.5 μmol/L imatinib for 4 hours, and the difference had statistical significance (P=0.022). These findings indicate that the co-culture with the BMSCs from patients with chronic myeloid leukemia can mediate K562 cels resistance to imatinib, which may be related to that the co-culture with BMSCs and exposure to imatinib can induce the K562 cels to express CXCR4. But the mechanism needs in-depth studies.

T-cell immune tolerance of HLA haploidentical donor induced by CTLA4Ig-modified bone marrow stromal cells / 中国组织工程研究

BACKGROUND:CTLA-4Ig as a tolerance-induction agent is a potential strategy in graft-versus-host disease prevention. OBJECTIVE:To investigate the efficacy of CTLA4Ig-gene-modified bone marrow stromal cels mediated by adenovirus to induce T-cel tolerance of haploidentical donors. METHODS: The bone marrow stromal cels isolated culture from the bone marrow of HLA haploidentical donors were transfected by recombinant adenovirus encoding CTLA4IgcDNA (AdCTLA4Ig) at a multiplicity of infection=50 for 72 hours. The expression rate and the location of CTLA4Ig in the transfected cels were detected by fluorescence microscope after immunofluorescence staining. CTLA4Ig-modified bone marrow stromal cels (2×104, 4×104and 8×104) were respectively co-cultured with 105 T cels from the peripheral blood of HLA haploidentical donors and 105 peripheral blood mononuclear cels from recipients. The proliferative inhibition rate was determined by MTT assay, and the level of interleukin-2 in the supernatant was detected by ELISA. The bone marrow mononuclear cels (1×105/wel) were co-cultured with CTLA4Ig-modified bone marrow stromal cel layers constructed in 6-wel plates. The number of bone marrow mononuclear cels and colony-forming unit-granulocyte macrophages were calculated after 5-day culture. RESULTS AND CONCLUSION: The expression rate of CTLA4Ig at the multiplicity of infection=50 was as high as 85%, and the immunofluorescence signals of CTLA4Ig were distributed unevenly in the cytoplasm. The inhibition rates of 2×104, 4×104, and 8×104 CTLA4Ig-modified bone marrow stromal cels on proliferation of T cels were higher than that of untransfected cels. The levels of interluekin-2 in the corresponding cel groups were significantly lower than that in the untransfected cels (P 0.05). These findings indicate that CTLA4Ig-modified bone marrow stromal cels mediated by adenovirus can induce immune tolerance of T-lymphocyte from HLA haploidentical donors in vitro.

Study on the roles of SDF-1/CXCR4 axis in imatinib resistance of K562 cells co-cultured with the bone marrow stromal cells / 中国医师杂志

Objective To investigate influences of co-culture with the bone marrow stromal cells (BMSCs) on imatinib sensitivity,and the role of stromal cell-derived factor-1 (SDF-1)/chemokine receptor 4 (CXCR4) axis in imatinib resistance of K562 cells in the co-culture model.Methods The model was constructed by co-culturing K562 cells with BMSCs isolated and cultured from the patients with chronic myeloid leukemia.The apoptosis rate and the CXCR4 expressing rate of the K562 cells exposed to 0.5 μmol/L imatinib for 72 hours were detected by fluorescent-activated cell scanning (FACS) machine.The K562 cells were exposed to 0.5 μmol/L imatinib for 4 hours,and labelled by calckin-AM fluorescent labeling sytem.The adhesion rate of the K562 cells co-cultured with BMSCs for 24 hours was calculated with fluorescence intensity.The IC50 value of K562 cells exposed to imatinib was detected by methyl thiazolyl tetrazolium (MTT) assay while the SDF-1/CXCR4 axis was blocked by 10 μg/ml monoclonal antibody of CXCR4.Results The apoptosis rate of K562 cells exposed to 0.5 μmol/L imatinib for 72 hours in co-culture group and suspension culture group was (15.48 ±4.17) ％ and (32.01 ±6.83) ％,respectively.The apoptosis rates of K562 cells in the two groups were significantly different (t =5.587,P =0.001).For the co-culture group,the CXCR4 expressing rates of K562 cells unexposed and exposed to 0.5 μmol/L imatinib for 72 hours were (20.31 ± 3.76) ％ (suspension cultured:11.28％ ± 3.44％) and (53.64 ± 5.35) ％ (suspension cultured:25.34％ ± 3.21％),respectively.Those results showed that co-culture with BMSCs and exposure to imatinib induced the K562 cells to express CXCR4.The adhesion rates of the K562 cells to the BMSCs were elevated from (42.18 ± 6.17) ％ to (68.97 ± 11.08) ％ when the K562 cells were exposed to 0.5 μmol/L imatinib for 4 hours.The IC50 values of block group (the SDF-1/CXCR4 axis was blocked by 10 μg/ml monoclonal antibody of CXCR4) and unblock group were (0.68 ± 0.04) μmol/L and (1.27 ± 0.05) μmol/L,respectively.The IC50 values of two groups were significantly different(t =4.869,P =0.001).Conclusions The K562 cells co-cultured with the BMSCs from the patients with chronic myeloid leukemia can obtain resistance to imatinib,which was related with that co-culture with the BMSCs and exposure to imatinib can induce the K562 cells to express CXCR4.To a certain extent,the imatinib resistance mediated by co-culture with BMSCs can be reversed by monoclonal antibody of CXCR4.