Establishment and bioinformatics evaluation of the ethanol combined with palmitic acid-induced mouse hepatocyte AFLD model (the Hu-Qiu Model).

Chronic alcoholic liver disease has brought great harm to human health. Alcoholic fatty liver disease is the first stage in the progression of all chronic alcoholic liver diseases. At present, there is no cell model that fully matches the etiology (high-fat diet + alcohol) of human alcoholic fatty liver disease. We used 100 mM ethanol +6.25 µM PA to establish the ethanol combined with PA-induced mouse hepatocyte AFLD model (EP-AFLD hepatocyte model) and performed the RNA-seq transcriptome sequencing. Through bioinformatics analysis and comparison, we discovered that the EP-AFLD hepatocyte model was more suitable for studying the pathological mechanism of AFLD than the mouse AFLD hepatocyte model induced by ethanol alone. And through bioinformatics analysis, we further discovered that 77 genes from the differential expression gene set of EP-AFLD hepatocyte model were engaged in the pathological process of mouse AFLD and 40 genes were involved in the pathogenesis of both mouse AFLD and human AFLD. In this study, a novel mouse hepatocyte AFLD model was successfully established by combining ethanol and PA, which can be used to study the molecular mechanism of the pathogenesis of AFLD in mice or humans. This study will provide a brand-new in vitro experimental platform for the in-depth study of AFLD pathogenesis and the screening of AFLD therapeutic drugs.

Association of CYP24A1 with survival and drug resistance in clinical cancer patients: a meta-analysis.

BACKGROUND: Acquired chemo-drug resistance constantly led to the failure of chemotherapy for malignant cancers, consequently causing cancer relapse. Hence, identifying the biomarker of drug resistance is vital to improve the treatment efficacy in cancer. The clinical prognostic value of CYP24A1 remains inconclusive, hence we aim to evaluate the association between CYP24A1 and the drug resistance in cancer patients through a meta-analysis approach. METHOD: Relevant studies detecting the expression or SNP of CYP24A1 in cancer patients up till May 2022 were systematically searched in four common scientific databases including PubMed, EMBASE, Cochrane library and ISI Web of Science. The pooled hazard ratios (HRs) indicating the ratio of hazard rate of survival time between CYP24A1high population vs CYP24A1low population were calculated. The pooled HRs and odds ratios (ORs) with 95% confidence intervals (CIs) were used to explore the association between CYP24A1's expression or SNP with survival, metastasis, recurrence, and drug resistance in cancer patients. RESULT: Fifteen studies were included in the meta-analysis after an initial screening according to the inclusion and exclusion criteria. There was a total of 3784 patients pooled from all the included studies. Results indicated that higher expression or SNP of CYP24A1 was significantly correlated with shorter survival time with pooled HRs (95% CI) of 1.21 (1.12, 1.31), metastasis with pooled ORs (95% CI) of 1.81 (1.11, 2.96), recurrence with pooled ORs (95% CI) of 2.14 (1.45, 3.18) and drug resistance with pooled HRs (95% CI) of 1.42 (1.17, 1.68). In the subgroup analysis, cancer type, treatment, ethnicity, and detection approach for CYP24A1 did not affect the significance of the association between CYP24A1 expression and poor prognosis. CONCLUSION: Findings from our meta-analysis demonstrated that CYP24A1's expression or SNP was correlated with cancer progression and drug resistance. Therefore, CYP24A1 could be a potential molecular marker for cancer resistance.

Epiregulin increases stemness-associated genes expression and promotes chemoresistance of non-small cell lung cancer via ERK signaling.

BACKGROUND: Chemoresistance often causes the failure of treatment and death of patients with advanced non-small-cell lung cancer. However, there is still no resistance genes signature and available enriched signaling derived from a comprehensive RNA-Seq data analysis of lung cancer patients that could act as a therapeutic target to re-sensitize the acquired resistant cancer cells to chemo-drugs. Hence, in this study, we aimed to identify the resistance signature for clinical lung cancer patients and explore the regulatory mechanism. METHOD: Analysis of RNA-Seq data from clinical lung cancer patients was conducted in R studio to identify the resistance signature. The resistance signature was validated by survival time of lung cancer patients and qPCR in chemo-resistant cells. Cytokine application, small-interfering RNA and pharmacological inhibition approaches were applied to characterize the function and molecular mechanism of EREG and downstream signaling in chemoresistance regulation via stemness. RESULTS: The RTK and vitamin D signaling were enriched among resistance genes, where 6 genes were validated as resistance signature and associated with poor survival in patients. EREG/ERK signaling was activated by chemo-drugs in NSCLC cells. EREG protein promoted the NSCLC resistance to chemo-drugs by increasing stemness genes expression. Additionally, inhibition of EREG/ErbB had downregulated ERK signaling, resulting in decreased expression of stemness-associated genes and subsequently re-sensitized the resistant NSCLC cells and spheres to chemo-drugs. CONCLUSIONS: These findings revealed 6 resistance genes signature and proved that EREG/ErbB regulated the stemness to maintain chemoresistance of NSCLC via ERK signaling. Therefore, targeting EREG/ErbB might significantly and effectively resolve the chemoresistance issue.

The effectiveness of the problem-based learning in medical cell biology education: A systematic meta-analysis.

BACKGROUND: Problem-based learning (PBL) was widely adopted in medical cell biology education for Chinese student; however, there was no systematic analysis to prove PBL was much more effective than lecture-based learning (LBL). Our aim is to evaluate the effectiveness of PBL on cell biology curriculum compared with LBL. METHOD: We systematically searched the publications related to PBL teaching approach in cell biology curriculum for medical education from databases until to February 2021. Pooled standard mean differences (SMDs) and risk ratios with their 95% confidence intervals were used to assess the effectiveness of PBL and the satisfaction of students to PBL compared to LBL in meta-analysis. The heterogeneity of the included studies was assessed by statistical I2 of heterogeneity. Meta-regression and subgroup analysis were performed to analyze the source of heterogeneity. Funnel plots and Egger tests were performed to assess publication bias. RESULT: After initial searching and selection, 9 studies were included for meta-analysis. All of these 9 studies were in high quality. The SMDs (95% confidence intervals) of total examination scores and comprehensive examination scores between PBL and LBL curriculum in cell biology teaching was calculated to be 0.89 (0.52, 1.26) and 0.53 (0.29, 0.78). Meanwhile, the risk ratios of the satisfaction of PBL vs LBL were calculated to be 1.18 (0.96, 1.46). However, there was a heterogeneity among the pooled SMDs of 10 studies with I2 = 89.7%, P < .001. The factors including the different teachers, the similar or same examination paper and over 100 student numbers among PBL and LBL groups raised the heterogeneity in the pooled SMDs. There is no publication bias in these 10 publications after Egger and Begg test. CONCLUSION: The result indicated PBL was better than LBL in improvement of examination scores and comprehensive examination scores in cell biology curriculum to some extent. However, the satisfaction of students to PBL and LBL had no difference. The factors, including the different teachers, the similar or same examination papers and over 100 student numbers, affected the effectiveness of PBL and raised the heterogeneity of the pooled SMDs.

Association of SMAD4 loss with drug resistance in clinical cancer patients: A systematic meta-analysis.

BACKGROUND: Drug resistance frequently led to the failure of chemotherapy for malignant cancers, hence causing cancer relapse. Thus, understanding mechanism of drug resistance in cancer is vital to improve the treatment efficacy. Here, we aim to evaluate the association between SMAD4 expression and the drug resistance in cancers by performing a meta-analysis. METHOD: Relevant studies detecting SMAD4 expression in cancer patients treated with chemo-drugs up till December 2020 were systematically searched in four common scientific databases using selected keywords. The pooled hazard ratio (HR) was the ratio of hazard rate between SMAD4neg population vs SMAD4pos population. The HRs and risk ratios (RRs) with 95% confidence intervals (CIs) were used to explore the association between SMAD4 expression losses with drug resistance in cancers. RESULT: After an initial screening according to the inclusion and exclusion criteria, eleven studies were included in the meta-analysis. There were a total of 2092 patients from all the included studies in this analysis. Results obtained indicated that loss of SMAD4 expression was significantly correlated with drug resistance with pooled HRs (95% CI) of 1.23 (1.01-1.45), metastasis with pooled RRs (95% CI) of 1.10 (0.97-1.25) and recurrence with pooled RRs (95% CI) of 1.32 (1.06-1.64). In the subgroup analysis, cancer type, drug type, sample size and antibody brand did not affect the significance of association between loss of SMAD4 expression and drug resistance. In addition, there was no evidence of publication bias as suggested by Begg's test. CONCLUSION: Findings from our meta-analysis demonstrated that loss of SMAD4 expression was correlated with drug resistance, metastasis and recurrence. Therefore, SMAD4 expression could be potentially used as a molecular marker for cancer resistance.

Analyzing the potential therapeutic mechanism of Huashi Baidu Decoction on severe COVID-19 through integrating network pharmacological methods.

BACKGROUND AND AIM: Huashi Baidu Decoction (HSBD) is a novel complex prescription which has positive effects on severe COVID-19. This study was aimed to discover key Chinese materia medica, main active compounds, hub therapeutic target proteins and core signal pathways in the potential therapeutic mechanism of HSBD on severe COVID-19 through integrating network pharmacological methods. EXPERIMENTAL PROCEDURE: TCMSP, TCMID and STITCH databases were used to screen out active compounds and target proteins of HSBD. GeneCards database was used to screen out disease genes of severe COVID-19. The potential therapeutic targets of HSBD on severe COVID-19 were used to construct protein-protein interaction network through STRING database and the hub target proteins were discovered. Next, GO and KEGG enrichment analysis were carried out to discover core signal pathways. Finally, the network diagram of "Chinese materia medica-active compounds-therapeutic target proteins" was built, then key Chinese materia medica and main active compounds were selected. RESULTS AND CONCLUSION: HSBD might treat severe COVID-19 through 45 potential target genes, among them, there were 13 hub target genes: RELA, TNF, IL6, IL1B, MAPK14, TP53, CXCL8, MAPK3, MAPK1, IL4, MAPK8, CASP8, STAT1. Meanswhile, GO_BiologicalProcess and KEGG signaling pathways analysis results showed that the core signal pathways were inflammation and immune regulation pathways. Finally, 4 key Chinese materia medica and 11 main active compounds were discovered in the HSBD. In conclusion, the therapeutic mechanism of HSBD on severe COVID-19 might involve its pharmacological effects of anti-inflammation and immune regulation via acting on 45 disease-related proteins of severe COVID-19. TAXONOMY CLASSIFICATION BY EVISE: Viral Pneumonia, COVID-19, Acute Respiratory Distress Syndrome, Septic Shock, Chinese Herbal Medicine.

The Study of Yin-Chen-Hao-Tang Preventing and Treating Alcoholic Fatty Liver Disease through PPAR Signaling Pathway Based on Network Pharmacology and RNA-Seq Transcriptomics.

BACKGROUND: Alcoholic fatty liver disease (AFLD) is the first stage of the alcoholic liver disease course. Yin-Chen-Hao-Tang (YCHT) has a good clinical effect on the treatment of AFLD, but its molecular mechanism has not been elucidated. In this study, we tried to explore the molecular mechanism of YCHT in improving hepatocyte steatosis in AFLD mice through network pharmacology and RNA sequencing (RNA-Seq) transcriptomics. METHODS: Network pharmacological methods were used to analyze the potential therapeutic signaling pathways and targets of YCHT on AFLD. Then, the AFLD mice model was induced and YCHT was administered concurrently. Liver injury was measured by serum alanine aminotransferase (ALT) activity and liver tissue H&E staining, and liver steatosis was determined by serum triglyceride (TG) level and liver tissue Oil Red staining. The molecular mechanism of YCHT on prevention and treatment of mice AFLD was investigated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differential expression genes data obtained by liver tissue RNA-Seq. Finally, ethanol-induced AFLD AML12 hepatocyte model was established, YCHT with or without PPARα agonist pemafibrate or PPARγ inhibitor GW9662 was administered, Nile Red fluorescent staining was used to evaluate steatosis levels in AML12 hepatocytes, and qRT-PCR was used to detect PPARα and PPARγ gene expression. RESULTS: The results of network pharmacology analysis showed that YCHT may exert its pharmacological effect on AFLD through 312 potential targets which are involved in many signaling pathways including the PPAR signaling pathway. AFLD mice experiments results showed that YCHT markedly decreased mice serum ALT activity and serum TG levels. YCHT also significantly improved alcohol-induced hepatic injury and steatosis in mice livers. Furthermore, KEGG pathway enrichment results of RNA-Seq showed that the PPAR signaling pathway should be the most relevant pathway of YCHT in the prevention and treatment of AFLD. AFLD hepatocyte model experiment results showed that YCHT could remarkably reduce hepatocyte steatosis through reducing PPARγ expression and increasing PPARα expression. CONCLUSIONS: Our study discovered that PPARγ and PPARα are the key targets and the PPAR signaling pathway is the main signaling pathway for YCHT to prevent and treat AFLD.

Total extract of Xin Jia Xuan Bai Cheng Qi decoction inhibits pulmonary fibrosis via the TGF-ß/Smad signaling pathways in vivo and in vitro.

PURPOSE: Pulmonary fibrosis (PF) is a common clinical disease, which results in serious respiratory impairment. Xin Jia Xuan Bai Cheng Qi Decoction (XJXBCQ) is a traditional prescription commonly used in treating lung diseases. We investigate the effect of XJXBCQ against PF and its mechanism via the regulation of TGF-ß1/Smad in vitro and in vivo. MATERIALS AND METHODS: XJXBCQ was first extracted and probed for chemical characterization. An PF model in vitro and in vivo was established in rats and in MRC-5 cells. In bleomycin (BLM)-induced rats model, lung function such as peak expiratory flow (PEF), minute ventilation (MV) and hydroxyproline (HYP) were measured; histopathological changes of lung tissue and TGF-ß1 in peripheral blood of rats were detected. TGF-ß receptor, Smad2 and its phosphorylation expression were tested by Western blot assay in rats model. Then the effects of XJXBCQ on TGF-ß1/Smad signal pathway were assessed by Western blot analysis in vitro, and IL-17A and IL-25 levels were evaluated by ELISA in vivo. RESULTS: Our results showed that XJXBCQ significantly enhanced the lung functions, such as PEF, MV and HYP, by reducing the expression level of lung inflammatory cytokine and the content and fibrosis of lung collagen. Moreover, XJXBCQ effectively inhibited TGF-ß1, Smad2 and its phosphorylation expression, and the activation of Smad7 in vitro and in vivo. Furthermore, XJXBCQ had an inhibitory effect on the α-smooth muscle actin (α-SMA) and fibronectin (Fn) in vitro and downregulated IL-17A and IL-25 by inhibiting the activation of TGF-ß1/Smad signaling pathway in vitro and in vivo. Further, XJXBCQ effectively inhibitied ventilation volume and peak expiratory content remodeling and hydroxyproline content through inhibition of TGF-ßRⅡ, Smad2 and its phosphorylation expression, and activation of Smad7 in vivo. CONCLUSION: XJXBCQ extract had an anti-PF effect in vitro and in vivo, which could be attributed to the inhibition of the expression of p-Smad2 and increase in the expression of Smad7 by regulating the TGF-ß1/Smad activity.

High mobility group box-1 release from H2O2-injured hepatocytes due to sirt1 functional inhibition.

BACKGROUND: High mobility group box-1 (HMGB1), recognized as a representative of damage-associated molecular patterns, is released during cell injury/death, triggering the inflammatory response and ultimately resulting in tissue damage. Dozens of studies have shown that HMGB1 is involved in certain diseases, but the details on how injured hepatocytes release HMGB1 need to be elicited. AIM: To reveal HMGB1 release mechanism in hepatocytes undergoing oxidative stress. METHODS: C57BL6/J male mice were fed a high-fat diet for 12 wk plus a single binge of ethanol to induce severe steatohepatitis. Hepatocytes treated with H2O2 were used to establish an in vitro model. Serum alanine aminotransferase, liver H2O2 content and catalase activity, lactate dehydrogenase and 8-hydroxy-2-deoxyguanosine content, nicotinamide adenine dinucleotide (NAD+) levels, and Sirtuin 1 (Sirt1) activity were detected by spectrophotometry. HMGB1 release was measured by enzyme linked immunosorbent assay. HMGB1 translocation was observed by immunohistochemistry/immunofluorescence or Western blot. Relative mRNA levels were assayed by qPCR and protein expression was detected by Western blot. Acetylated HMGB1 and poly(ADP-ribose)polymerase 1 (Parp1) were analyzed by Immunoprecipitation. RESULTS: When hepatocytes were damaged, HMGB1 translocated from the nucleus to the cytoplasm because of its hyperacetylation and was passively released outside both in vivo and in vitro. After treatment with Sirt1-siRNA or Sirt1 inhibitor (EX527), the hyperacetylated HMGB1 in hepatocytes increased, and Sirt1 activity inhibited by H2O2 could be reversed by Parp1 inhibitor (DIQ). Parp1 and Sirt1 are two NAD+-dependent enzymes which play major roles in the decision of a cell to live or die in the context of stress . We showed that NAD+ depletion attributed to Parp1 activation after DNA damage was caused by oxidative stress in hepatocytes and resulted in Sirt1 activity inhibition. On the contrary, Sirt1 suppressed Parp1 by negatively regulating its gene expression and deacetylation. CONCLUSION: The functional inhibition between Parp1 and Sirt1 leads to HMGB1 hyperacetylation, which leads to its translocation from the nucleus to the cytoplasm and finally outside the cell.

HMGB1 release by H2O2-induced hepatocytes is regulated through calcium overload and 58-F interference.

HMGB1 is passively released by injured or dying cells and aggravates inflammatory processes. The release of HMGB1 and calcium overload have each been reported to be important mediators of H2O2-induced injury. However, a potential connection between these two processes remains to be elucidated. In the present study, we employed H2O2-induced hepatocytes to investigate how calcium overload takes place during cellular injury and how the extracellular release of HMGB1 is regulated by this overload. In addition, we investigated the use of 58-F, a flavanone extracted from Ophiopogon japonicus, as a potential therapeutic drug. We show that the PLCγ1-IP3R-SOC signalling pathway participates in the H2O2-induced disturbance of calcium homoeostasis and leads to calcium overload in hepatocytes. After a rise in intracellular calcium, two calcium-dependent enzymes, PKCα and CaMKIV, are activated and translocated from the cytoplasm to the nucleus to modify HMGB1 phosphorylation. In turn, this promotes HMGB1 translocation from the nucleus to the cytoplasm and subsequent extracellular release. 58-F effectively rescued the hepatocytes by suppressing the PLCγ1-IP3R-SOC signalling pathway and decreasing the calcium concentration in cells, thus reducing HMGB1 release.

58-F, a flavanone from Ophiopogon japonicus, prevents hepatocyte death by decreasing lysosomal membrane permeability.

Lysosome membrane permeabilization (LMP) has been implicated in cell death. In the present study, we investigated the relationship between cell death and H2O2-/CCl4-induced LMP in hepatocytes in vitro and following acute liver injury in vivo. The key finding was that H2O2 triggered LMP by oxidative stress, as evidenced by a suppression of LAMP1 expression, a reduction in LysoTracker Green and AO staining, and the leakage of proton and cathepsin B/D from the lysosome to the cytoplasm, resulting in cell death. CCl4 also triggered hepatocyte death by decreasing lysosome LAMP1 expression and by inducing the accumulation of products of peroxidative lipids and oxidized proteins. Furthermore, a novel compound 5,8-dimethoxy-6-methyl-7-hydroxy-3-3(2-hydroxy-4-methoxybenzyl) chroman-4-one (58-F) was extracted from Ophiopogon japonicus and served as a potential therapeutic drug. In vivo and in vitro results showed that 58-F effectively rescued hepatocytes by decreasing LMP and by inducing lysosomal enzyme translocation to the cytosol.

Panax Notoginseng flower saponins (PNFS) inhibit LPS-stimulated NO overproduction and iNOS gene overexpression via the suppression of TLR4-mediated MAPK/NF-kappa B signaling pathways in RAW264.7 macrophages.

BACKGROUND: Panax Notoginseng flower saponins (PNFS) are the main active component of Panax notoginseng (Burk) F. H. Chen flower bud (PNF) and possess significant anti-inflammatory efficacy. This study aims to explore the mechanisms underlying PNFS' antiflammatory action in RAW264.7 macrophages. METHODS: A cell counting kit-8 assay was used to determine the viability of RAW264.7 macrophages. Anti-inflammation effects of PNFS in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were measured based on the detection of nitric oxide (NO) overproduction (Griess method, DAF-FM DA fluorescence assay and NO2 (-) scavenging assay), and interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha gene overexpression (real-time PCR and ELISA). Inducible nitric oxide synthase (iNOS) gene overexpression was determined by real-time PCR and western blotting. iNOS enzyme activity was also assayed. The mechanisms underlying the suppression of iNOS gene overexpression by PNFS were explored using real-time PCR and western blotting to assess mRNA and protein levels of components of the Toll-like receptor 4 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-kappa B) signaling pathways. RESULTS: PNFS (50, 100, 200 µg/mL) significantly reduced LPS-induced overproduction of NO (P < 0.001, P < 0.001, P < 0.001) and IL-6 (P = 0.103, P < 0.001, P < 0.001), but did not affect TNF-alpha overproduction. PNFS (50, 100, 200 µg/mL) also markedly decreased LPS-activated iNOS (P < 0.001, P < 0.001, P < 0.001) and TLR4 gene overexpression (P = 0.858, P = 0.046, P = 0.005). Furthermore, treatment with PNFS (200 µg/mL) suppressed the phosphorylation of MAPKs including P38 (P = 0.001), c-Jun N-terminal kinase (JNK) (P = 0.036) and extracellular-signal regulated kinase (ERK) 1/2 (P = 0.021). PNFS (200 µg/mL) inhibited the activation of the NF-kappa B signaling pathway by preventing the phosphorylation of inhibitor of NF-kappa B alpha (I-kappa B alpha) (P = 0.004) and P65 (P = 0.023), but PNFS (200 µg/mL) could not activate the LPS-induced PI3K-Akt signaling pathway. CONCLUSIONS: PNFS significantly down-regulated iNOS gene overexpression and thereby decreased NO overproduction via the inhibition of TLR4-mediated MAPK/NF-kappa B signaling pathways, but not the PI3K/Akt signaling pathway.

Effect of Yiguanjian decoction on cell differentiation and proliferation in CCl4-treated mice.

AIM: To investigate the cellular mechanisms of action of Yiguanjian (YGJ) decoction in treatment of chronic hepatic injury. METHODS: One group of mice was irradiated, and received enhanced green fluorescent protein (EGFP)-positive bone marrow transplants followed by 13 wk of CCl4 injection and 6 wk of oral YGJ administration. A second group of Institute for Cancer Research mice was treated with 13 wk of CCl4 injection and 6 wk of oral YGJ administration. Liver function, histological changes in the liver, and Hyp content were analyzed. The expression of α-smooth muscle actin (α-SMA), F4/80, albumin (Alb), EGFP, mitogen-activated protein kinase-2 (PKM2), Ki-67, α fetoprotein (AFP), monocyte chemotaxis protein-1 and CC chemokine receptor 2 were assayed. RESULTS: As hepatic damage progressed, EGFP-positive marrow cells migrated into the liver and were mainly distributed along the fibrous septa. They showed a conspicuous coexpression of EGFP with α-SMA and F4/80 but no coexpression with Alb. Moreover, the expression of PKM2, AFP and Ki-67 was enhanced dynamically and steadily over the course of liver injury. YGJ abrogated the increases in the number of bone marrow-derived fibrogenic cells in the liver, inhibited expression of both progenitor and mature hepatocyte markers, and reduced fibrogenesis. CONCLUSION: YGJ decoction improves liver fibrosis by inhibiting the migration of bone marrow cells into the liver as well as inhibiting their differentiation and suppressing the proliferation of both progenitors and hepatocytes in the injured liver.

[The analysis of the influencing factors of mortality rate in the transplantation of mouse bone marrow].

The purpose of the study was to investigate the influencing factors of mortality rate in the bone marrow transplantation in mice. The recipient mice receiving whole-body irradiation of gamma-ray were infused with the same strain of bone marrow cells or the mixture of the bone marrow cells and splenocytes respectively. Experiments were carried out in four batches, with different strains of mice used, respectively. The manifestations and the appearance of graft-versus-host disease (GVHD) were observed, as well as the mortality rate within 35 d of the transplantation in the recipient mice. The mortality rate of the first group of recipient mice was the lowest, the mortality rate of the second group of recipient mice was the highest and the obvious GVHD performance was observed before death. In the third group, the mortality rate declined and there was statistical significance compared to that of the second group. The mortality rate of the fourth group of mice was higher than that of the third group, but still lower than that of the second group of mice and there is a statistical significance. This evidence suggested that mouse genetic purity, splenocytes, the ratio of the bone marrow cells and splenocytes and the week-old of the mouse could be the important influencing factors of the mortality rate in mouse bone marrow transplantation.