Establishment and Evaluation of Intestinal Injury Model of Mouse Acute Graft Versus Host Disease Based on An Organoid Technology / 中国实验血液学杂志

OBJECTIVE@#To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology.@*METHODS@#20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×107/mice, n=20) and spleen nucleated cells (5×106/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5+ and Clu+ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings.@*RESULTS@#The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5+ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu+ cells were observed in the 5% group. The Lgr5+ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu+ cells in the 20% group significantly increased.@*CONCLUSION@#The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.

Effects of different nitrogen sources on accumulation of active components in hairy roots of Salvia miltiorrhiza and Salvia castanea f. tomentosa / 中草药

Objective: Nitrogen is an important element affecting the accumulation of effective components in Chinese medicinal materials. The purpose of this study was to investigate effects of different nitrogen sources on the growth and active components accumulation of hairy roots of Salvia miltiorrhiza and Salvia castanea f. tomentosa. Methods: The hairy roots of S. miltiorrhiza and S. castanea f. tomentosa were treated with ammonium nitrate, hydrolyzed milk protein, peptone, beef extract, casein and yeast extract, respectively. The growth of hairy roots and the accumulation of active components were analyzed. Results: Ammonium nitrate was the most beneficial to the growth of the two kinds of hairy roots. Hydrolyzed milk protein significantly promoted the accumulation of salvianolic acids, compared with ammonium nitrate, the contents of rosmarinic acid and salvianolic acid B in S. miltiorrhiza were respectively increased by 2.94 times and 3.27 times, and the contents of rosmarinic acid and salvianolic acid B in S. castanea f. tomentosa were respectively increased by 13.74 times and 2.01 times. Yeast extract had the most significant effect on the accumulation of dihydrotanshinone I and cryptotanshinone in two kinds of hairy roots. Hydrolyzed milk protein significantly promoted the accumulation of tanshinone IIA in the hairy roots of S. miltiorrhiza, while beef extract had the most significant effect on the accumulation of tanshinone IIA in S. castanea f. tomentosa. Conclusion: Ammonium nitrate was the best nitrogen source for the growth of two kinds of hairy roots, and hydrolyzed milk protein was the best nitrogen source for salvianolic acids accumulation. The effects of different nitrogen sources on four kinds of tanshinones were different, and the responses of S. miltiorrhiza and S. castanea f. tomentosa to different nitrogen sources were also different. This study not only has certain guiding significance for large-scale cultivation of hairy roots of S. miltiorrhiza and industrialized production of active components, but also provides a reference for the development and utilization of S. castanea f. tomentosa resources.

Effects of different carbon sources on growth and active component contents in Salvia miltiorrhiza and S. castanea f. tomentosa hairy roots / 中国中药杂志

Salvia miltiorrhiza(Sm) and Salvia castanea f. tomentosa(Sc) hairy roots were used as experimental materials to study the effects of six different carbon sources, galactose, fructose, lactose, glucose, arabinose and sucrose(control), on fresh weight, dry weight, contents and yields of salvianolic acids and tanshinones. The results showed that galactose was most beneficial to the growth of two kinds of hairy roots, while lactose and arabinose were not conducive to their growth. As for Sm hairy roots, fructose significantly promoted the accumulation of salvianolic acid B, and the content increased by 5.801 times and 10.151 times compared with the control group, respectively. Glucose significantly promoted the accumulation of salvianolic acids. The content and yield of rosmarinic acid were 7.674 times and 9.260 times of that of the control group, and the content and yield of salvianolic acid B were 5.532 times and 6.675 times of the control group. For the hairy roots of Sc, galactose significantly increased the content and yield of rosmarinic acid, reaching 7.820 times and 9.944 times of the control group, respectively. Fructose promoted the increase of the content and yield of cryptotanshinone, reaching 9.242 times and 6.609 times of the control group, respectively. The study confirmed the optimal carbon source for the hairy root culture of Sm and Sc, and provided theoretical guidance for large-scale production of Sm drug-derived components and the utilization of Sc.

Analysis of Clinical Pathological Features and Prognosis in Young Patients with Diffuse Large B Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To explore the clinical pathological features of the patients with diffuse large B cell lymphoma (DLBCL) and their prognostic factors.@*METHODS@#The prognosis of the clinical pathological features and their influence on prognosis of 177 patients diagnosed as DLBCL at the first visit from January 2013 to May 2017 in our hospital were analyzed retrospectively.@*RESULTS@#The univariate analysis showed that overall survival (OS) and progression-free survival (PFS) were associated with later Ann Arbor stage (Ⅲ-Ⅳ) ( P＜0.01, P＜0.05), high performance status (ECOG score 2-4) (P＜0.01, P＜0.05), extranodal involvement ＞1 (P＜0.01, P＜0.05), elevated LDH level (P＜0.01, P＜0.05). B symptom (P＜0.05) and elevated β2-MG level (P＜0.05) also influenced OS. COX multivariate analysis showed that the elevated β2-MG level (P＜0.05) and later stage (Ⅲ-Ⅳ) (P＜0.05) have an independent influence on OS, later stage (Ⅲ-Ⅳ) (P＜0.05) also independently influenced PFS. The patients with high aaIPI score (2-3) and bone marrow involvement before treatment had poor OS (P＜0.01, P＜0.01) and PFS (P＜0.05, P＜0.01).@*CONCLUSION@#Elevated β2-MG level can independently influence OS, and later stage (Ⅲ-Ⅳ) can independently influence both OS and PFS. High aaIPI score (2-3) and bone marrow involvement before treatment have an inferior influence on OS and PFS.

Application of Chimeric Antigen Receptor-Modified NK Cells in Multiple Myeloma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the killing effect of CAR (CD138-CD28-CD3ζ)-NK cells on myeloma cells through construction of CAR(CD138-CD28-CD3)-NK cells.</p><p><b>METHODS</b>The antiCD138scFv-CD28-CD3 zeta plasmid pcDNA3.1 was constructed, which then together with 3 plasmid lentiviral packaging system were transfected into 293T cells, the virus was collected. Furthermore, in order to get the stably transfected cell line, the NK92MI cell line was infected by the virus, then the positive cells were screened by puromycin. The expression of the CARNK cells were verified by RT-PCR and Western blot. At last the ability of secreting cytokine CD107a was detected by flow cytometry, and the statistical analysis was carried out to verify the anti-myeloma effect of CAR-NK cells.</p><p><b>RESULTS</b>Gene fragment of the CAR(antiCD138scFv-CD28-CD3ζ) was constructed successfully by gene engineering technique in vitro, and the gene sequence was verified to be correct by sequencing. By virus packaging technology, the virus expressing the protein of the CAR was obtained. PCR and Western blot verified the expression of CAR fusion protein on the sufurce of NK cells. The cell killing experiment confirmed that the CAR-NK cells possessed the ability to secrete cytokine CD107a superior to control cells and showed the obvious killing effect on multiple myeloma cells.</p><p><b>CONCLUSION</b>The CAR can be constructed in vitro, and express on NK92 cells. The CAR-NK cells can kill the multiple myeloma cells expressing CD138 antigen, thereby plays an antimyeloma effect.</p>

Establishment of A20 Murine B Lymphoma Transplantation Model Labeled with Luciferase / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish the animal model of luciferase-transfected A20 murine B cell lymphoma, so as to provide experimental tools to explore the effect of graft versus tumor.</p><p><b>METHODS</b>Luciferase- labeled A20 cells were cloned with puromycin selection. Transfected A20 cells and CBL/6 bone marrow were inoculated into the irradiated BALB/c mice by injection in tailvein to establish the transplantation model. The bioluminescent imaging technique was used to monitor the tumor growth, and then the survival, body weight, tumor formation and pathological characteristics of target organs were observed.</p><p><b>RESULTS</b>A20 cell line stably expressing luciferase gene was successfully obtained. The the bioluminicent imaging found that the tumor luminescence could be observed on day 8 of A20 cell inoculation, and the mean fluorescent intensity was increased along with the tumor growth. Compared with the BMT group, the survival rate and body weight of BMT+A20-Lucmice were decreased significantly. General anatomy showed the tumor mainly formed in the liver and spleen.</p><p><b>CONCLUSION</b>A mouse transplantation model with luciferase- transfected A20 cells has been successfully established, thus laying a foundation for investigation of graft-versus-tumor.</p>

Advances of Diagnostic Biomarkers on Acute Graft Versus Host Disease -Review / 中国实验血液学杂志

Acute graft-versus-host disease (aGVHD) is one of the main complications after allogeneic hematopoietic stem cell transplantation (HCT), resulting in considerable morbidity and mortality. Diagnosis of aGVHD is typically based on clinical presentation and confirmed by biopsy. In recent years, aGVHD researches have been focused on diagnostic biomarkers, and several microRNAs, cytokines and immunoregulated cells have been dicovered currently. This review summarizes the advances on aGVHD biomarkers.

Treatment of Chronic Graft versus Host Disease -Review / 中国实验血液学杂志

Chronic graft versus host disease (cGVHD) is the major reason of late non-relapse related death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The therapeutic regimen based on glucocorticoids remains as the standard initial treatment of cGVHD. But due to the significant side effects and unsatisfactory outcomes, particularly for patients with steroid-refractory disease, the more effective and less toxic therapies should be needed. Abnormal regulation for the stable state of T lymphocytes and B lymphocytes for allo-HSCT patients is the current focus of studying cGVHD pathogenesis. This article summarizes the treatment of cGVHD targeted to pathogenic factors and signaling pathways in pathogenetic process.

Therapeutic Efficacy Analysis of Allogeneic Peripheral Blood Hematopoietic Stem Cell Transplantation for 14 Adult Patients with T Lymphoblastic Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic efficacy of allogeneic peripheral blood hematopoietic stem cell transpdantation (allo-HSCT) for T lymphoblastic lymphoma (T-LBL).</p><p><b>METHODS</b>The clinical data of 14 adult patients with T-LBL treated with allo-HSCT were collected, the hematopoietic reconstruction, survival and relapse, as well as overall survival (OS) rate, event-free survival (EFS) rate of 1, 3 and 5 years were analysed retrospectively.</p><p><b>RESULTS</b>All the patients were engrafted with neutrophil successfully, the median time of absolute neutrophil count >0.5 × 10(9)/L was 13 (10-19) d; 13 patients were engrafted with platelets successfully, the median time of Plt count >20 × 10(9)/L was 17 (12-62) days. The acute GVHD occurred in 6 patients, but among them only 1 case with 3 grade of aGVHD; out of 14 patients, 5 developed chronic GVHD. The transplant-related mortality at 100 days was 7.1% (1/14), mainly from coronary heart disease and pulmonary infection. The median follow-up time was 26.5 months, the estimated 1, 3 and 5 year OS rate was 85.7%, 47.6% and 38.1%, respectively, and estimated 1, 3 year EFS rate was 85.7%, 34.4% and 34.1%, respectively. The relapse rate was 42.8% (6/14) and the median relapse time was 22.5% months after transplantation. Up to now, 7 patients still survive, 1 patient out of them have survived for 103 months.</p><p><b>CONCLUSION</b>The allo-HSCT is a safe and effective method for treatment of T-LBL.</p>

Effect of rhG-CSF Mobilization on S1P5 Expression in T Lymphocyte Subsets of Allo-HSCT Donors / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) mobilization on S1P5 expression in T lymphocyte subsets of allo-HSCT donors.</p><p><b>METHODS</b>The peripheral blood was collected from 10 allo-hematopoietic stem cell transplantation (allo-HSCT) donors before and after mobilization with rhG-CSF for 4 days. The flow cytometry was used to detect S1P5 expression in T lymphocyte subsets.</p><p><b>RESULTS</b>There was no S1P5 expression on the surface of T-lymphocytes both before and after rhG-CSF mobilization. After fixation with permeabilization agent, S1P5 expression could be detected in lymphocytes after rhG-CSF mobilization, which indicates S1P5 may be located in cells. Compared with level before rhG-CSF mobilization, S1P5 expression was significantly increased in T lymphocyte subsets after rhG-CSF mobilization, CD3(+)T cells (57.92±2.32)% vs (7.94±1.47)%(P<0.05）, CD4(+)T cells (72.58±1.73)% vs （5.48±0.82）%(P<0.05）, CD8(+)T cells（51.79±3.57）% vs （6.46±1.01）%（P<0.05）,CD3-/CD56(+)NK cells（40.00±1.47）% vs（4.97±0.74）%（P<0.05）. The up-regulated level of S1P5 expression in CD4(+)T cells was most high(P<0.05).</p><p><b>CONCLUSION</b>S1P5 expression significantly increases in T lymphocyte subsets after rhG-CSF mobilization, and the up-regulated level of S1P5 expression in CD4(+)T cells is the most high.</p>

Application of CRISPR/Cas9 Gene Editing Technique to Establish S1PR5 Gene Knockout Mice / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish the S1PR5 gene knockout mouse model by using CRISPR/Cas9 gene editing technique so as to provide the tool for studying the regulating role of sphingosine-1-phosphate receptor 5 (S1PR5) in allogeneic hematopoietic stem cell transplantation.</p><p><b>METHODS</b>Single guide RNA (sgRNA) plasmids against the exon 3 of S1PR5 were designed and constructed. Then the sgRNA and hCas9 were transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA were mixed and microinjected into fertilized eggs of C57BL/6 mice. T7E1 digestion and gene sequencing were used to detect the mutations of S1PR5. Quantitative PCR (qPCR) and Western blot were used to detect the expression of S1PR5.</p><p><b>RESULTS</b>Finally 2 kinds of F2 generation of homozygous S1PR5 deficent mice (S1PR5-170/-170 mice and S1PR5-215/-215 mice) were gained, and in these 2 model mice the S1PR5 did not express at mRNA and protein levels.</p><p><b>CONCLUSION</b>A mouse model with S1PR5 dificiency has been successfully established, which shall lay a foundation for future investigation of S1PR5.</p>

Expression Profile of lncRNA NONHSAT040475 in Peripheral Blood Leukocytes of Healthy Persons / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression profile of lncRNA NONHSAT040475 in peripheral blood leukocytes of healthy persons.</p><p><b>METHODS</b>The peripheral blood mononuclear cells were collected from 10 healthy volunteers, the CD3(+) T cells,CD19(+) B cells,CD56(+) NK cells and granulocytes were purified and sorted by flow cytometry. Then, the expression of lncRNA NONHSAT040475 in peripheral blood leukocyte subsets was detected by real-time quantitative PCR.</p><p><b>RESULTS</b>the expression levels of lncRNA NONHSAT040475 were various in lymphocyte subsets, its expression in B lymphocytes was significantly higher, as compared with T lymphocytes, while the expressions of lncRNA NONHSAT040475 in NK cells and granulocytes were relative low（P<0.01）.</p><p><b>CONCLUSION</b>lncRNA NONHSAT040475 is widely expressed in human peripheral blood leukocytes, and mainly expressed in B lymphocytes, therefore, laying the foundation for further study of B lymphocyte-related diseases. This study has brought a new opportunity for diagnosing and illustrating the molecular mechanism of hematologic disorder or autoimmune disease.</p>