Chemical constituents and their bioactivities from seeds of Clausena lansium / 中草药

Objective To study the chemical constituents and their biological activities of the seeds of Clausena lansium. Methods The compounds were isolated by various column chromatographic techniques including silica gel, Sephadex LH-20, semi-preparative HPLC, et al., and their structures were identified through a combined analysis of physicochemical properties, as well as NMR and MS data. The in vitro α-glucosidase inhibitory activity and nematicidal activity against Panagrellus redivivusl were screened by PNPG and Berman funnel methods, respectively. Results Eleven compounds were isolated and identified as (4R*,6R*)-6-hydroxypiperitone (1), (4S*,6R*)-6-hydroxypiperitone (2), (1S*,2S*,4R*)-1-methyl-4-(prop-l-en-2-yl) cyclohexane-1,2-diol (3), subamone (4), methyl (1R*,2R*,2’Z)-2-(5’-hydroxy-pent-2’-enyl)-3-oxo-cyclopentane acetate (5), 5-hydroxy-4-phenyl-5H-furan-2-one (6), loliolide (7), xylogranatinin (8), 2,6-dihydroxyhumula-3(12),7(13),9(E)-triene (9), xanthoxol (10), and ligballinol (11). Conclusion Compounds 1-8 are isolated from the genus for the first time, and compound 9 is first isolated from this species. Compound 8 showed strong α-glucosidase inhibitory activity, and compound 5 exhibited potent nematicidal activity.

Study on sesquiterpenes from agarwood originating from Gyrinops salicifolia / 中国中药杂志

Two sesquiterpenes were isolated from the agarwood originating from Gyrinops salicifolia with various chromatographic techniques, and their structures were determined as 12-hydroxy-dihydrocyperolone(1) and(rel)-4β,5β,7β-eremophil-9-en-12,8α-olide(2), through a combined analysis of physicochemical properties and spectroscopic evidence. Compound 1 was a new compound. Compound 2 showed cytotoxicities against K562 and BEL-7401 cell lines, with IC_(50) values of(17.85±0.04) and(21.82±0.07) mg·L~(-1), respectively [taxol as positive control, with IC_(50) values of(1.97±0.11) and(6.31±0.08) mg·L~(-1)].

The wound healing potential of collagen peptides derived from the jellyfish Rhopilema esculentum / 中华创伤杂志(英文版)

PURPOSE@#Wound represents a major health challenge as they consume a large amount of healthcare resources to improve patient's quality of life. Many scientific studies have been conducted in search of ideal biomaterials with wound-healing activity for clinical use and collagen has been proven to be a suitable candidate biomaterial. This study intended to investigate the wound healing activity of collagen peptides derived from jellyfish following oral administration.@*METHODS@#In this study, collagen was extracted from the jellyfish--Rhopilema esculentum using 1% pepsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fourier transform infrared (FTIR) were used to identify and determine the molecular weight of the jellyfish collagen. Collagenase II, papain and alkaline proteinase were used to breakdown jellyfish collagen into collagen peptides. Wound scratch assay (in vitro) was done to determine migration potential of human umbilical vein endothelial cells (HUVEC) covering the artificial wound created on the cell monolayer following treatment with collagen peptides. In vivo studies were conducted to determine the effects of collagen peptides on wound healing by examining wound contraction, re-epithelialization, tissue regeneration and collagen deposition on the wounded skin of mice. Confidence level (p < 0.05) was considered significant using GraphPad Prism software.@*RESULTS@#The yield of collagen was 4.31%. The SDS-PAGE and FTIR showed that extracted collagen from jellyfish was type I. Enzymatic hydrolysis of this collagen using collagenase II produced collagen peptides (CP) and hydrolysis with alkaline proteinase/papain resulted into collagen peptides (CP). Tricine SDS-PAGE revealed that collagen peptides consisted of protein fragments with molecular weight <25 kDa. Wound scratch assay showed that there were significant effects on the scratch closure on cells treated with collagen peptides at a concentration of 6.25 μg/mL for 48 h as compared to the vehicle treated cells. Overall treatment with collagen peptide on mice with full thickness excised wounds had a positive result in wound contraction as compared with the control. Histological assessment of peptides treated mice models showed remarkable sign of re-epithelialization, tissue regeneration and increased collagen deposition. Immunohistochemistry of the skin sections showed a significant increase in β-fibroblast growth factor (β-FGF) and the transforming growth factor-β (TGF-β) expression on collagen peptides treated group.@*CONCLUSION@#Collagen peptides derived from the jellyfish-Rhopilema esculentum can accelerate the wound healing process thus could be a therapeutic potential product that may be beneficial in wound clinics in the future.

A new eudesmane type sesquiterpene from cultivated Clerodendranthus spicatus in Hainan / 中国中药杂志

Six compounds were isolated from the aerial part of cultivated Clerodendranthus spicatus in Hainan with various chromatographic techniques,and their structures were determined as:1-dehydroxy-1-oxo-rupestrinol(1),N-trans-feruloyltyramine(2),methyl 3,4-dihydroxyphenyllactate(3),caffein acid(4),methyl caffeate(5) and ethyl caffeate(6),via analysis of physicochemical properties and spectroscopic evidence.Compound 1 was a new compound,while compounds 2 and 3 were isolated from C.spicatus for the first time.Biological activity results showed that compounds 2-4 exhibited α-glucosidase inhibitory activity with different inhibition ratio.

The intervention of ginkgo biloba extract on monocrotaline-induced right ventricular hypertrophy in rats and its mechanism / 中国应用生理学杂志

OBJECTIVE@#To study the protective effects of ginkgo biloba extract on the right ventricular hypertrophy.@*METHODS@#Seventy-two SD male rats were randomly divided into 3 groups: control group(CON), monocrotaline-induced right ventricular hypertrophy group (MCT) and ginkgo biloba extract treated group (EGB) (n=24 in each group). Group MCT and group EGB were intraperitoneally injected with 2%MCT at the dose of 60 mg /kg on the first day. From the second day, group MCT was injected with 2 ml 0.9% sodium chloride, and 60 mg/kg ginkgo leaf extract was administered to the stomach in group EGB. The control group was injected with 2 ml 0.9% sodium chloride on the first day. After 3 weeks, in each group,cardiac hemodynamic changes were measured, heart weight index was calculated, and myocardial pathological changes were observed by HE staining. The expression of TRPC6 was detected by real-time polymerase chain reaction (real-time -PCR) and Western blot.@*RESULTS@#Compared with the control group, the right ventricular systolic pressure (RVSP) was increased significantly in the MCT group(P＜0.01), the maximum or decline rate of descent (RV ±dp/dt) of the right ventricle pressure was increased significantly(P＜0.01), while the EGB group had the same trend as all the indexes in the group MCT, but the amplitude of all indicators in group EGB were decreased significantly than those of group MCT(P＜0.01), and the right ventricular hypertrophy index (RVMI) in group EGB was significantly lower than that in group MCT(P＜0.01).Group MCT showed typical myocardial hypertrophy performance by HE staining, and the right ventricular myocytes in group EGB were significantly improved than that in group MCT, and the mRNA and protein expression levels of TRPC6 in the right ventricle of group MCT and group EGB were increased(P＜0.01), while the EGB group was significantly lower than that of the MCT group(P＜0.01).@*CONCLUSION@#Ginkgo biloba extract may inhibit the signal pathway of CaN / NFAT in cardiac myocytes by reducing the expression of TRPC6 protein, and then play an early protective effect on myocardial hypertrophy.

A new sesquiterpene from Chinese agarwood induced by artificial holing / 中国中药杂志

In order to study the chemical constituents of n-butanol fraction of ethanol extract from Chinese agarwood induced by artificial holing, various chromatographic techniques were carried out to isolate compounds, and the structures of compounds were determined through a combined analysis of physicochemical properties and spectroscopic evidence. Seven compounds were obtained and identified as selina-3,11-dien-9,15-diol (1), aquilarone D (2), 5α,6β,7α,8β-tetrahydroxy-2-[2-(2-hydroxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone (3), 6,7-dimethoxy-2-[2-(4-methoxyphenyl)ethyl]chromone (4), syringin (5), methyl (Z)-p-coumarate (6), and 4'-methoxycinnamic acid (7), among which compound 1 was a new compound and compounds 5-7 were isolated from agarwood for the first time. The bioactivity assay results concluded that compounds 6 and 7 showed certain nematicidal activity against Panagrellus redivivus, and compounds 4, 6 and 7 exhibited cytotoxicity against BEL-7402, SGC-7901 and A549 carcinoma cell lines.

Morinda Officinalis How improves cellphone radiation-induced abnormality of LH and LHR in male rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Morina Officinalis How (MOH) on the abnormal levels of serum luteotrophic hormone (LH) and LH receptor (LHR) in the testis tissue induced by cellphone radiation (CPR) in rats.</p><p><b>METHODS</b>Fifty adult male SD rats were randomly divided into five groups of equal number: sham CPR, untreated CPR, negative double distilled water (DDW) control, aqueous MOH extract, and alcohol MOH extract. All the animals were exposed to mobile phone radiation except those of the sham CPR group. Then, the rats of the latter two groups were treated intragastrically with MOH at 20 g per kg of the body weight per day in water and alcohol, respectively. After 2. weeks of treatment, all the rats were sacrificed for measurement of the levels of serum LH and LHR in the testis tissue.</p><p><b>RESULTS</b>The levels of serum LH and LHR were 30.15 ± 8.71 and 33.28 ± 6.61 in the aqueous MOH group and 0.96 ± 0.06 and 0.94 ± 0.08 in the alcohol MOH group, both significantly decreased as compared with the negative DDW controls (P < 0.05), but with no remarkable difference between the two MOH groups (P > 0.05).</p><p><b>CONCLUSION</b>MOH can improve CPR-induced abnormality of LH and LHR in adult male rats.</p>

A new diterpene from the stems of Trigonostemon heterophyllus / 中国天然药物

AIM@#To investigate the chemical constituents in the stems of Trigonostemon heterophyllus.@*METHOD@#The chemical constituents were isolated by column chromatography on silica gel, Rp-18, and Sephadex LH-20, and their structures were elucidated on the basis of spectroscopic analysis.@*RESULTS@#Three compounds were isolated and identified as a new diterpene, trigonoheterene B (1), together with two known compounds, trigonostemone (2) and trigonochinene B (3).@*CONCLUSION@#Compound 1 is new. Compounds 2 and 3 showed antibacterial activities.

Effects of chronic hypoxia on left and right ventricular function and the expression of cardiac TRPC channels in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the effects of chronic hypoxia on left and right ventricular function and the expression of cardiac transient receptor potential canonical (TRPC) channels in rats.</p><p><b>METHODS</b>Forty eight SD male rats were randomly divided into control group (CON) and chronic hypoxic pulmonary hypertension model group (CH) (n = 24). In CH group, rats were exposed in chronic hypoxia environment (10% +/- 0.2% O2) to induce myocardial hypertrophy. After 3 weeks, mean systemic arterial pressure (mSAP), right ventricular systolic pressure (RVSP), left ventricular systolic pressure (LVSP), left or right ventricular pressure maximum rate of rise (LV/RV + dp/dt(max)), left or right ventricular pressure maximum rate of descent (LV/RV-dp/dt(max)), right ventricular hypertrophy index (RVMI) and left ventricular hypertrophy index (LVMI) were measured. Left and right ventricular myocardium tissue sections were stained by HE and observed under light microscope. Real-time polymerase chain reaction (real-time-PCR) and Western blot were performed to detect the expression of TRPC subfamily.</p><p><b>RESULTS</b>RVSP, RVMI, RV + dp/dt(max) and RV-dp/dt(max) were markedly elevated in CH group (P < 0.01) in comparison to CON group. LVMI was markedly reduced in CH group in comparison to CON group (P < 0.01). LVSP, LV + dp/dt(max) and LV- dp/dt(max) had no significant changes in CH group in comparison to CON group. Right ventricular myocardial cells of CH group became thick, the nuclei stained deeply, the shape of nuclei became not regularity. Left ventricular myocardial fibers did not change significantly. There was significant difference in the levels of mRNA and protein of TRPC1 between CON and CH groups.</p><p><b>CONCLUSION</b>For three weeks exposed to chronic hypoxia induced right ventricular hypertrophy specifically, raised the mRNA and protein expression of TRPC1 on right ventricular myocardial cells . TRPC1 might be involved in the development of cardiac hypertrophy.</p>

Verification of a method with low usage amount of arsenic trioxide for determining urinary iodine by As (Ⅲ)-Ce4+ catalytic spectrophotometry using ammonium persulfate digestion / 中国地方病学杂志

Objective To verify a new revised method with low usage amount of arsenic trioxide for determining urinary iodine by As(Ⅲ)-Ce4+ catalytic spectrophotometry using ammonium persulfate digestion.Methods The standard curve linearity,sample detection limit,precision and accuracy of determining urinary iodine of this modified method were verified according to Determination Methods of Chemicals in Biological Materials.Results The linear correlative coefficients of the 0-300 μg/L range and 300-1200 μg/L range calibration curve were-0.9998--1.0000(n =6) and-0.9998--1.0000,respectively.The detection limit for iodine was 1.3 μg/L.The relative standard deviations were 1.5％ (1.1/71.3)-2.5％ (6.2/244.9) when measuring 3 urine samples with iodine concentration of 71.3-244.9 μg/L,and 0.6％(2.4/388.5)-1.7％(17.3/1018.0) when measuring 3 urine samples with iodine concentration of 388.5-1018.0 μg/L,respectively(n =6).The test results of the four urinary iodine national standard materials with iodine concentration of 73.0,206.0,556.0 and 883.0 μg,/L were all within the given value range and the average value relative deviation was 1.8％ (1.3/73.0),0.4％ (0.8/206.0),0.2％ (1.0/556.0) and-1.6％(-13.7/883.0),respectively (n =6).The average recovery was 98.8％ with a range of 93.2％ (186.3/200.0)-103.4％(51.7/50.0) when measuring 3 urine samples with iodine concentration of 64.6-144.9 μg/L and 3 urine samples with iodine concentration of 346.8-574.4 μg/L,respectively.Conclusions This new modified method greatly reduces the amount of waste containing arsenic,and can directly take urine samples with high iodine concentration to digest and determine without dilution.It is performed with good standard linear curve,better precision and high accuracy,and in line with the analysis of biological samples requirements.

Analysis of the county-level evaluation results after reaching the goal of eliminating iodine deficiency disorders in Longyan city, Fujian province in 2009 / 中国地方病学杂志

Objective To understand the current situation of iodine deficient disorder(IDD) 10 years after achieving the stage goal of eliminating IDD in Longyan city and to evaluate the effect of prevention and treatment measures, and to provide the basis for the development of control strategies. Methods There were 7 counties in the city, and each county(city, district) was as a unit to carry out the inspection for organization and leadership,iodine salt management, monitoring and control, health education (referred to as the four management indicators)according to "The County-Level Assessment and Evaluation Implementation Detailed Rules of Realizing the Goal to Eliminate IDD in Fujian Province". According to the east, west, south, north and middle positions in each county,a village and a primary school were selected. Forty 8 to 10 year-old students in each school were randomly selected to check thyroid and among them 20 students were collected urine samples to determine urinary iodine. Nine townships were selected in the 7 counties of the city and among which 4 administrative villages were selected in each township. Eight edible salt samples from each household in each administrative village were collected to test salt iodine. Goiter was examined by palpation, the level of urinary iodine was examined by arsenic and cerium spectrophotometry, salt iodine was detected by direct titration. Results The average score of the four management indicators was 94.1 in Longyan city. The adjusted goiter rate of children aged 8 - 10 years old was 1.9%. The median of urinary iodine was 278.6 μg/L, among which less than 100 μg/L accounted for 4.57%(32/700), 100 -＜ 200 μg/L accounted for 24.00%(168/700), 200 - ＜ 300 μg/L accounted for 25.29%(177/700), and higher than 300 μg/L accounted for 46.14%(323/700). The using rate of qualified iodized salt was 98.86%. The coverage rate of iodized salt was 99.50%, the qualified rate of iodized salt was 99.35%, and the rate of non-iodized salt was 0.50%. All the indicators had reached the national standard to eliminate IDD. Conclusions After achieving the stage goal of eliminating IDD, the disease is stable and the effect of control measures are significantly. But the iodine provided has a trend of more than suitable. Therefore, it is reasonable to reduce the current salt iodine content.

Effects of human aorta-gonad-mesonephros region stromal cells on inducing differentiation of murine embryonic stem cells into hematopoietic stem/progenitor cells in vitro / 中国实验血液学杂志

This study was aimed to investigate the effect of human aorta-gonad-mesonephros (AGM) region stromal cells on differentiation of murine embryonic stem cells (ESC) into hematopoietic stem cells (HSC) in vitro and to clarify their effect mechanism. E14 murine ESC were induced into embryo body (EB) firstly. Then the EB cells were further co-cultured with the stromal cells from human AGM region, fetal liver (FL) or bone marrow (BM) in Transwell non-contact system. According to the different culture methods, the EB cells were divided into 6 groups including EB control group, AGM group, FL group, BM group, AGM + FL group and AGM + BM group. The induced cells derived from EB were collected for Sca-1(+)/c-Kit(+) cells analysis by flow cytometry and colony forming unit (CFU) assay. The results showed that Sca-1(+)/c-Kit(+) cell proportion of EB cells significantly increased after being induced by different stromal cells (p < 0.01). The AGM + FL group had most Sca-1(+)/c-Kit(+) cells for the positive cell proportion reached (21.96 ± 2.54) % (p < 0.01). The Sca-1(+)/c-Kit(+) cell proportion of AGM + BM group was much high than that of BM group too (p < 0.01). The EB control group showed CFU amount less than any other groups, while the CFU amount of AGM + FL, AGM + BM groups were higher, especially in the AGM + FL group (p < 0.01). It is concluded that the human AGM region stromal cells may help to maintain certain number of primitive HSC with high proliferation potential. Human AGM region, FL or BM stromal cells, applied in sequential orders, can significantly expand in vitro the primitive hematopoietic stem/progenitor cells derived from ESC.

Sequentially inducting murine embryonic stem cells into hematopoietic stem cells in vitro by hematopoietic development procedure for reconstitution of hematopoiesis in vivo / 中国实验血液学杂志

This study was purposed to directly induce murine embryonic stem cells (ESC) into hematopoietic stem cells (HSC) by simulating the spatial and temporal hematopoietic microenvironment changes in embryonic development, and to investigate the function of in vivo hematopoietic reconstitution of these HSC. E14 ESC were induced into embryoid body (EB) firstly. Then the cells from EB were further co-cultured with human aorta-gonad-mesonephros (AGM) region, fetal liver (FL) and bone marrow (BM) stromal cells in Transwell non-contact system in sequential orders. After 6 days of each co-cultured stage, the induced cells derived from EB were collected to analyze the Sca-1(+)c-Kit(+) cells by flow cytometry, check teratoma formation and transplant to BALB/C female mice exposed to lethal dose (60)Co γ-ray. The recipient mice were divided randomly into 5 groups: AGM, AGM + FL, AGM + FL + BM, irradiation control and normal control groups. The survival rates, hematopoietic reconstitution and engraftment of donor cells in the different groups were monitored. The results showed that Sca-1(+)c-Kit(+) cell level in EB cells co-cultured with human AGM region and FL stromal cells reached to peak value (21.96 ± 2.54)%. Teratomas could be found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region stromal cells, while there was no teratoma in the mice after subcutaneous injection of EB cells induced by human AGM region and FL stromal cells. The recipients in AGM group and irradiation control group all died. The survival rate was 77.8% in AGM+FL group, and 66.7% in AGM+FL+BM group. The peripheral blood cell count was near normal at day 21 after transplantation, and Sry gene copies from donor could be detected in recipient mice of these two groups. It is concluded that sequentially inductive system with feeder cells from human AGM region, fetal liver and bone marrow simulating embryonic defined hematopoiesis procedures can enhance the directed differentiation of ESC into HSC which can safely reconstitute hematopoiesis in vivo.

Outcome analysis of surveillance on iodine deficiency disorders in Longyan City in Fujian Province in the year 2006 and 2007 / 中国地方病学杂志

Objective To understand the current situation of iodine deficiency diserders(IDD) in Longyan City and to evaluate the effect of prevention and control measures of IDD in order to provide evidence for formulating prevention and control tactics. Methods During the year of 2006 and 2007, the 30 primary schools were screened by population proportion survey(PPS) from the 7 counties of Longyan City. Forty children aged 8-10 years in each school were randomly selected as a group to examine thyroid, and 7 children in each group were selected to measure the urine iodine and the salt iodine at the same time. The goiter rote, the median urinary iodine, the consumption rate of qualified iodized salt, the iodine salt coverage rate, the rate of qualified iodized salt and the non-iodized rate were detected. Results The goiter rate of children aged 8-10 years old in Longyan City was 0.94%(79/8438). The median urinary iodine was 259.12 μg/L. The consumption rate of qualified iodized salt was 97.86% (1462/1494). The iodine salt coverage rate was 99.46%(1486/1494). The rate of qualified iodized salt was 98.38 (1462/1486), and the non-iodized rate was 0.54% (8/1494). Conclusions All indicators have reached the national standard of eliminating IDD in Longyan City.

In vitro suppressive effect of angelica polysaccharide on human cytomegalovirus-induced apoptosis via direct infection in CHRF-288-11 cells / 中国实验血液学杂志

The objective of study was to investigate the in vitro suppressive effect of angelica polysaccharide (APS) on human cytomegalovirus-induced apoptosis via direct infection in CHRF-288-11 cells. HCMV AD169 directly infected CHRF-288-11 were cultured in vitro, APS in different doses were added on day 3 after the infection of virus. Cells of every group were collected at different time points. HCMV DNA of cells were detected by using polymerase chain reaction and the apoptotic cells were examined by using Hoechst staining, MTT assay, DNA fragmentation assay and flow cytometry. The results showed that the APS to some extent inhibited the apoptosis of CHRF cells infected by HCMV in vitro in a dose-dependent manner. The expression of HCMV IEA in CHRF-288-11 cells was found by PCR amplification. Morphology observation, flow cytometry assay and DNA fragmentation assay revealed the existence of apoptosis. With the dose decrease of APS added to the infected CHRF cells, the percentage of apoptotic cells increased. It is concluded that the HCMV AD169 can infect CHRF cells directly in vitro and decrease cell viability. HCMV AD169 infection increases the apoptosis of CHRF cells in time-dependent manner. When APS was added to the CHRF cells infected by HCMV AD169 in vitro, the viability of CHRF cells increase, which indicated that APS to some extent protects the CHRF cells infected by HCMV. APS suppresses the cytomegalovirus-induced apoptosis in CHRF cells directly infected in vitro in dose-dependent manner.

Effect of intra-bone marrow infusion of allogeneic mesenchymal stem cells on reconstruction of marrow mesenchymal stem cells in rat HSCT models / 中国实验血液学杂志

This study was aimed to investigate the effect of intra-bone marrow (IBM) injection of allogeneic mesenchymal stem cells (MSCs) on reconstruction of bone marrow MSCs (BM-MSCs) in rats that received hematopoietic stem cell transplantation (HSCT), and to detect the donor MSCs in the hosts for clarifying the effect mechanism of donor MSCs. Wistar female rats conditioned with lethal dose 60Co gamma-ray irradiation were co-transplanted with F344 female fetal and neonatal peripheral blood (FNPB) and BrdU-labeled MSCs separated from bone marrow mononuclear cells of F344 male rats. The donor MSCs were infused by IBM injection in bilateral tibia or intravenous injection (IV), while the FNPB were all via IBM route. The survival rate, engraftment level of HSCs and recovery of BM-MSCs of recipients were monitored. The ratio of BrdU-labeled MSCs in recipient rats was calculated by immunofluorescence assay (IFA) and the Y chromosomes were examined by PCR. The results showed that the recipient rats of the two co-transplantation groups were all alive at day 60 after transplantation. There was no significant difference between these two groups on the survival rates or the engraftment levels of HSCs, but each of them was much better than that of the FNPB group. At day 30 after transplantation, the proliferation ability of recipients' BM-MSCs was still below normal, while that of the FNPB (IBM)+MSC (IBM) group was the best of all the experiment groups (p<0.01). At 60 days, the donor MSCs coexisted with host MSCs in only a few recipient rats examined by IFA, while the Y chromosomes could be detected in all the recipient rats in the two cotransplantation groups. It is concluded that the infusion of allogeneic MSCs can accelerate the recovery of HSCT recipients' BM-MSCs. The IBM route is safe and more effective than intravenous infusion.

Isolation and culture of human embryonic AGM derived HSPCs in hematopoietic culture systems created by AGM stromal cells / 中国实验血液学杂志

This study was purposed to isolate human embryonic AGM derived HSPCs and investigate the effect of AGM stromal cells on AGM-derived HSPCs. Immunohistochemical sections of human AGM tissue were investigated for CD34, Flk-1 and VEGF expression. Human AGM-derived single cells were isolated and seeded onto pre-treated feeder of human AGM stromal cells (hAGMS3 and hAGMS4) by direct contact and non-contact co-culture in Transwell culture system. Growth characteristics of HSPCs with cobblestone area-forming cells (CAFCs) were observed and number of cobblestone area (CA) was counted. Indirect immunofluorescent assay was used to detect CD34 and Flk-1 expression on the surface of suspended cells as well as CAFCs in contact co-culture system. The cells after culture for 2 weeks were collected from both contact and non-contact co-culture systems for CFU assay. The result showed that hematopoietic cells in AGM tissue expressed CD34 and Flk-1. Both of the hematopoietic culture systems could produce CFCs. Nevertheless, direct contact co-culture produced CD34(+)Flk-1(+) CAFC and more CFUs than those from indirect non-contact culture (hAGMS3 system: 1647 +/- 194 vs 389 +/- 31, p < 0.05; hAGMS4 system: 1586 +/- 75 vs 432 +/- 35, p < 0.05). It is concluded that there were CD34(+)Flk-1(+) HSCs in human embryonic AGM region. The hematopoietic co-culture systems composed of AGM-derived HSPCs and AGM stromal cells are successfully established, both direct contact and Transwell non-contact co-culture can expand AGM-derived definitive HSPCs. Cell-cell contact between AGM-derived HSPCs and AGM stromal cells are of most importance to maintain and expand AGM-HSPCs.

BMP-4 and VEGF promote development of hematopoietic stem cells during the embryoid body formation from embryonic stem cells / 中国实验血液学杂志

The objective of this study was to explore the effects of BMP-4 and VEGF on the development of primary hematopoietic stem cells during the differentiation of embryonic stem cells (ESCs) into embryoid body (EB). Murine E14 ESCs were seeded into semisolid methylcellulose-based medium for EB formation. According to added or not cytokines, experiments were divided into: (1) group of spontaneous differentiation without cytokine as control; (2) group of BMP-4 in different concentrations (0, 5, 15, 25 and 50 ng/ml); (3) group of BMP-4 combined with VEGF; (4) group of VEGF alone. EBs were collected on days 3, 6, 9, 12, 15, and the proportion of Flk-1(+) cells were assayed by flow cytometry. The results showed that in the different BMP-4 concentration groups, the proportions of Flk-1(+) cells were significantly different, and it reached the peak values in 25 ng/ml BMP-4 group as 6.51 +/- 1.02% at day 3 and 7.70 +/- 1.12% at day 6 respectively, which were statistically higher than those in control group without-BMP-4 and in 5 ng/ml BMP-4 group (p < 0.05). When BMP-4 was used in combination with VEGF, Flk-1(+) cells went to peak proportion value at day 9 as 27.53 +/- 8.14%, which was statistically higher than that in spontaneous differentiation group as 8.77 +/- 2.35% (p < 0.05) and VEGF treatment group as 11.21 +/- 2.23% (p < 0.05). It is concluded that BMP-4 in combination with VEGF can promote Flk-1(+) cells genesis during EB formation in vitro, which provides experimental evidence for researches on directed differentiation of ESCs into hematopoietic stem cells simulating the microenvironment in vivo.

Comparison of efficacies of allogeneic hematopoietic stem cell transplantations between different routes of administration in mice / 中国实验血液学杂志

When hematopoietic stem cells (HSCs) were administrated by intravenous infusion (IV), most of them were trapped in some nonhematopoietic organs as like lungs that had abundant blood capillaries. Only a small fraction of injected cells could home to the bone marrow, which reduced the engraftment of HSCs. The purpose of intra-bone marrow (IBM) transplantation was to facilitate the homing of HSCs directly. Based on the established murine model for allogeneic umbilical cord blood transplantation (UCBT) by IBM injection, the objective of this study was to compare the distribution of fetal and neonatal peripheral blood (FNPB) mononuclear cells (MNC) in vivo and the efficacy of HSCT by different routes of administration in mice. BALB/c recipient mice exposed to sublethal dose 60Co gamma-ray were transplanted with FNPBMNCs from C57BL/6 mice. Recipient mice were divided into six groups at random: unilateral-IBM group; bilateral-IBM group; IV group; bilateral-IBM + IV group; irradiated control group and normal group. The distribution of CFSE-labeled FNPBMNCs in the recipients was observed in frozen sections of different organs or by flow cytometry. The survival rate, engraftment level, recovery of hematopoietic function and GVHD of recipient mice were studied. The results showed that infused by IBM route, FNPBMNCs mainly accumulated in the bone marrow (BM) cavity of the injected side tibia. Some of them could enter the BM of noninjected bones via blood circulation and few were trapped in the lung. Though same amount of FNPBMNCs were injected into recipient mice of unilateral and bilateral-IBM group, less cells could leak into peripheral blood or other tissues when transplanted by bilateral-IBM route. Therefore, in term of accelerating hemopoietic recovery, the injection of IBM route was better than IV route, especially bilateral IBM injection of HSCs, which neared the normal level of peripheral blood cells and colony-forming units of bone marrow nucleated cells at day 21 after transplantation, followed by unilateral-IBM group and bilateral-IBM + IV group. The percentages of H-2Db cell subsets in the three IBM groups were much higher than that in IV group. There was no significant difference of the engraftment level in the injected side tibia between the unilateral and bilateral-IBM group. When secondary transplantation was performed, the engraftment level in bilateral-IBM group was still much higher than that in IV group. At day 90, the survival rates of IBM groups were all > or = 80%, while that of IV group was only 50%. It is concluded that bilateral-IBM route can facilitate the homing of more HSCs, accelerate the engraftment of HSCs and hematopoietic reconstitution, which promoted the efficacy of IBM-HSCT.

Expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros region / 中国实验血液学杂志

The objective of this study was to investigate the expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros (AGM) region. Stromal cells derived from human AGM region (hAGM S1-S5) and fibroblasts derived from human fetal trunk (hFT) were cultured in vitro. RT-PCR was used to analyze the expression of BMP-4 in hAGM S1-S5 stromal cells at mRNA level. And BMP-4 level was detected in the supernatant of hAGM S1-S5 stromal cells by ELISA assay. hFT cells were used as control group. The results showed that the heterogenous hAGM S1-S5 stromal cells displyed shapes of fibroblast-like and endothelial-like cells. hAGM S1-S5 stromal cells expressed BMP-4 mRNA, but fetal trunk fibroblasts (hFT) did not express BMP-4 mRNA. In the supernatant of hAGM S1-S5 cells, BMP-4 could be detected by ELISA assay ana its levels were statistically higher than that in hFT group (p < 0.05), while there was no significant difference between groups of hAGM S1-S5 (p > 0.05). It is concluded that human AGM-derived stromal cells in vitro express BMP-4, and the establishment of a new culture system based on the feeder cells of AGM stroma would promote the differention of embryonic stem cells into hematopoietic stem cells at a high proportion.