Superparamagnetic Iron Oxide Labeling of Neural Stem Cells and 4.7T MRI Tracking in vivo and in vitro

Neural stem cells were labeled with superparamagnetic iron oxide (SPIO) and tracked by MRI in vitro and in vivo after implantation. Rat neural stem cells were labeled with SPIO combined with PLL by the means of receptor-mediated endocytosis. Prussian blue staining and electron mi-croscopy were conducted to identify the iron particles in these neural stem cells. SPIO-labeled cells were tracked by 4.7T MRI in vivo and in vitro after implantation. The subjects were divided into 5 groups, including 5×105 labeled cells cultured for one day after labeling, 5×105 same phase unla-beled cells, cell culture medium with 25 μg Fe/mL SPIO, cell culture medium without SPIO and dis-tilled water. MRI scanning sequences included T1WI, T2WI and T2WI. R2 and R2 of labeled cells were calculated. The results showed: (1) Neural stem cells could be labeled with SPIO and labeling efficiency was 100%. Prussian blue staining showed numerous blue-stained iron particles in the cyto-plasm; (2) The average percentage change of signal intensity of labeled cells on T1WI in 4.7T MRI was 24.06%, T2WI 50.66% and T2WI 53.70% respectively; (3) T2 of labeled cells and unlabeled cells in 4.7T MRI was 516 ms and 77 ms respectively, R2 was 1.94 s-1 and 12.98 s-1 respectively, and T2 was 109 ms and 22.9 ms, R2 was 9.17 s-1 and 43.67 s-1 respectively; (4) Remarkable low signal area on T2WI and T2WI could exist for nearly 7 weeks and then disappeared gradually in the left brain transplanted with labeled cells, however no signal change in the right brain implanted with unlabeled cells. It was concluded that neural stem cells could be labeled effectively with SPIO. R2 and R2 of labeled cells were increased obviously. MRI can be used to track labeled cells in vitro and in vivo.     

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-β<Subscript>2</Subscript><Emphasis Type="Italic">in vitro</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Summary Whether transforming growth factor-β₂ (TGF-β₂) induces apoptosis of human trabecular meshwork cells was investigatedin vitro. Cultured 3–5 passage human trabecular meshwork cells were treated with 0 (control). 0. 32. 1, 3. 2 ng/ml TGF-β₂ for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy. TUNEL technique and flow cytometry. The results showed character istic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshowork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44)%. (4.43±1.17)% and (9.60±2.05)% respectively with different concentrations [1 ng/ml (P<0.05), 3.2 ng/ml (P<0.01)] of TGF-β₂ with the difference being significant between experimental group and control group [(1.41±0.34)%]. It was concluded that TGF-β₂ can induce apoptosis of human trabecular meshwork, cellsin vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people. CAO Yang, male, born in 1972, M. D., Ph. D., Associate Professor This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

Inhibitory effect of extract of fungi of <Emphasis Type="Italic">Huaier</Emphasis> on hepatocellular carcinoma cells

This study investigated the inhibitory effect of the extract of fungi of Huaier （EFH） on the growth of hepatocellular carcinoma （HCC） cells. Hep-G2 cells, a human HCC cell line, were cultured in DMEM containing 10% fetal bovine serum and treated with EFH of different concentrations （1, 2, 4, 8 mg/mL） for 24, 48 and 72 h respectively. The apoptosis rate of the cells was flow cytomet-rically measured. Thirty-six tumor-bearing New Zealand rabbits were randomly divided into 3 groups group A （control group）, in which the rabbits were infused with 0.2 mL/kg normal saline via the hepatic artery; group B （transhepatic artery cbemoembolization [TACE] group）, in which the rabbits were given lipiodol at 0.2 mL/kg plus MMC at 0.5 mg/kg via the hepatic artery; group C （TACE ＋ EFH group ）, in which EFH （500 mg/kg） were orally administered after TACE. Two weeks after TACE, the rabbits were sacrificed and the implanted tumors were sampled. The tumor volume and the necrosis rate were determined. The tumor tissues were immunohistochemically detected for the expressions of factor Ⅷ, VEGF, P53, Bax and Bcl-2. The microvessel density （MVD） was calculated by counting the factor Ⅷ-positive endothelial cells. Our results showed that after treatment with EFH the apoptosis rate of Hep-G2 cells was enhanced in a concentration- and time-dependent manner. Two weeks after the treatment, the average tumor volume, the necrosis rate and the growth rate of the implanted tumor in group C were significantly different from those in groups A and B （P〈0.05）. MVD and VEGF expressions were significantly decreased in the group C when compared with those in groups B （P〈0.05 for all）. The Bax expression was weakest in group A and strongest in group C. The expressions of P53 and Bcl-2 were minimal in group C and maximal in group A. There were significant differences in the expressions of P53, Bax and Bcl-2 among the 3 groups （P〈0.05 for all） and there was significant difference between group B and group C （P〈0.05）. It was concluded that EFH could suppress not only the growth of HCC cells but also tumor angiogenesis and it can induce the apoptosis of HCC cells. EFH serves as an alternative for the treatment of HCC.     

Cloning and Identification of frc Gene from Oxalobacter Frmigenes

The cloning and identification of frc gene from Oxalobacter formigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacter formigenes was extracted. frc gene fragment was amplified by polymerase chain reaction (PCR) and linked with pEGFP-C1. The recombinant plasmid was designated pEGFP-frc and was identified by restriction-enzyme digestion and sequencing. Human embryo kidney 293 cells were transfected with pEGFP-frc, then RT-PCR and Western blotting were performed to detect the expression of frc gene. The length of frc gene was found to be 1287 bp, and the homology of nucleotides and amino-acid residue with the sequence in GenBank was 95.88% and 99.07%. Bright green fluorescent light could be observed in 293 cells transfected with the pEGFP-frc. frc mRNA and fusion protein FCoAT-EGFP were detected in the cells. It is concluded that frc gene cloned from the Oxalobacter formigenes in the intestines of Chi- nese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.     

HBsAg/HBsAb double positive hepatitis B virus infection model <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

The pathogenesis of HBsAg （＋）/HBsAb （＋） double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes Could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.     

Angiogenic Potency of Bone Marrow Stromal Cells Improved by ex Vivo Hypoxia Prestimulation

Summary: To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells(BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random.In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six-weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced by ex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved by ex vivo hypoxia prestimulation though the enhanced VEGF expression.     

Experimental Study on the Antibacterial Effect of Origanum Volatile Oil on Dysentery Bacilli In Vivo and In Vitro

To observe the germistatic and germicidal effects of origanum volatile oil (OVI) on the dysentery bacteria, the abdominal cavity of mice was infected with Shigella sonne (Sh. sonnei) and Shigella flexneri (Sh. flexneri) F2a. After OVI was given to the mice via gastric lavage, the effects of OVI on the infected mice were observed. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for dysentery bacteria were determined in vitro. The resuits showed that origanum volatile oil showed obvious protective effect on mice infected with Sh. sonnei and Sh. flexneri F2a. and it had germistatic and germicidal effects on dysentry bacteria. We are led to conclude that origanum volatile oil is an effective medicine against the infection of dysentery bacteria.     

Effects of Lutein on the Growth and Migration of Bovine Lens Epithelial Cells In Vitro

The effects of lutein on the growth and migration of bovine lens epithelial cells （BLECs） in vitro were observed in an attempt to find a drug that can prevent after-cataract. BLECs were cultured in vitro and different concentrations of lutein were added to the BLECs cultures of the second and third generations. The effects of lutein on the proliferation of BLECs in vitro were examined by the MTT method, and the migration of BLECs was evaluated by a scratch wound assay. The results showed that： （1） Lutein at concentrations of 1 to 16 μmol/L could inhibit the proliferation of BLECs in a dose-and time-dependent manner （P〈0.01）; （2） The migration of BLECs was evaluated by wound healing rate. As compared with the control group, the wound healing rate in the experimental groups was decreased from 0.672±0.164 to -0.234±0.144 and -0.597±0.063 （P〈0.01） at 1 and 2 μmol/L lutein, respectively. It was concluded that lutein at concentration of 〉1 μmol/L inhibited the proliferation and migration of BLECs in vitro, Lutein may become an effective drug to prevent after-cataract.     

Effects of carvedilol on cardiomyocyte apoptosis and gene expression<Emphasis Type="Italic">in vivo</Emphasis> after ischemia-reperfusion in rats

Summary The effects of carvedilol on cardiomyocyte apoptosis and expression of bcl-2, bax genes following ischemia (0.5 h) and reperfusion (48 h)in vivo and the possible biological mechanism of carvedilol inhibiting cardiomyocyte apoptosis were studied. The left anterior descending artery in Wistar rats were ligated to establish ischemia-reperfusion (I/R) models. The model animals were divided into two groups: I/R group, the model rats not subject to other treatments except ischemia-reperfusion (n=8): carvedilol-treated group (n=8), I/R model rats treated with carvedilol. Eight rats in the sham-operated group were subjected to only experimental open operation. The number of apoptotic cardiomyocyte was determined by TUNEL staining. Immunohistochemistry andin situ hybridization histochemistry (ISHH) were used to detect the expression of bcl-2 and bax genes. Image processing system was used to quantitatively dispose the positive metric substances of both immunohistochemistry and ISHH through the average optical density (OD) value. The results showed that the number of the apoptotic cells were 36.18±9 (I/R group), 0–1 (sham-operated group) and 9.5±3 (carvedilol-treated group) in each visual field respectively with the difference being very significant among the groups (P<0.001). The OD values of bcl-2 protein in sham-operated group, I/R group and carvedilol-treated group were 0.14±0.01, 0.08±0.02 and 0.15±0.03, respectively. The OD values of bcl-2 mRNA in sham-operated group, I/R group and carvedilol-treated group were 0.08±0.01, 0.06±0.01 and 0.09±0.01, respectively. There was no significant difference between carvedilol-treated group and I/R group (P>0.05). The OD values of bax protein in I/R group, sham-operated and carvedilol-treated-treated group were 0.13±0.02, 0.07±0.01, 0.06±0.01, respectively. There was very significant difference between carvedilol-treated group and I/R group (P<0.01). Bcl-2/bax ratio was 1.07±0.14 (I/R group), 1.28±0.16 (sham-operated group), 2.5±0.26 (carvedilol-treated group) respectively with the difference being very significant between carvedilol-treated group and I/R group (P<0.05). It was indicated that carvedilol could inhibit cardiomyocyte apoptosis following ischemia and reperfusion, which was related to the increased bcl-2/bax ratio due to inhibition of bax gene expression. ZENG Hesong, male, born in 1965, Associated Professor This project was supported by a grant from the National Natural Sciences Foundation of Hubei Province (No. 2000J050).     

In vitro Recombination and Identification of Mutated Fragment Corresponding to Regulation Region of mtrR Gene of Neisseria Gonorrhoeae

A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria Gonorrhoeae(NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene.According to the technique of gene splicing by overlap extension(SOEing),a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction(PCR).The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced.By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains,2 of these were not compatible completely.The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter.It can be confirmed that the fragments EF are the specifically designed mutant fragments.     

骨髓抑制性细胞在肝细胞癌患者外周血中的水平及其临床价值

 目的  筛选肝细胞癌(hepatocellular carcinoma,HCC)患者外周血骨髓抑制性细胞(myeloid-derived suppressor cells,MDSCs)并验证其抑制功能,同时检测外周血MDSCs水平。方法  收集2014年3月10月在复旦大学附属中山医院诊治的HCC患者60例、慢性肝炎患者25例和健康人50例。采用流式细胞仪方法检测HCC患者、慢性肝炎患者和健康人外周血的MDSCs水平,分析MDSCs在HCC患者、慢性肝炎患者和健康人之间的差异,并评价MDSCs在患者临床病理资料中的差异。结果   HCC患者外周血MDSCs水平显著高于慢性肝炎患者和健康人群,HCC患者外周血MDSCs水平与肿瘤包膜、微血管侵犯显著相关(P<0.05)。同时MDSCs具有抑制T细胞增殖和分泌的功能。结论  术前外周血MDSCs水平有助于提示HCC患者的病理情况,为HCC患者的治疗提供重要的辅助依据。     

Different optical properties between human hepatocellular carcinoma tissues and non-tumorous hepatic tissues <Emphasis Type="Italic">In Vitro</Emphasis>

There has been an ongoing search for clinically acceptable methods for the accurate,efficient and simple diagnosis and prognosis of hepatocellular carcinoma(HCC).Optical spectroscopy is a technique with potential clinical applications to diagnose cancer diseases.The purpose of this study was to obtain the optical properties of HCC tissues and non-tumorous hepatic tissues and identify the difference between them.A total of 55 tissue samples(HCC tissue,n=38;non-tumorous hepatic tissue,n=17) were surgically resected from patients with HCC.The optical parameters were measured in 10-nm steps using single-integrating-sphere system in the wavelength range of 400 to 1800 nm.It was found that the optical properties and their differences varied with the wavelength for the HCC tissue and the non-tumorous hepatic tissue in the entire wavelength range of research.The absorption coefficient of the HCC tissue(1.48±0.99,1.46±0.88,0.86±0.61,2.15±0.53,0.54±0.10,0.79±0.15 mm-1) was significantly lower than that of the non-tumorous hepatic tissue(2.79±1.73,3.13±1.47,3.06±2.79,2.57±0.55,0.62±0.10,0.93±0.16 mm-1) at wavelengths of 400,410,450,1450,1660 and 1800 nm,respectively(P<0.05).The reduced scattering coefficient of HCC tissue(5.28±1.70,4.91±1.54,1.26±0.35 mm-1) and non-tumorous hepatic tissue(8.14±3.70,9.27±3.08,2.55±0.57 mm-1) was significantly different at 460,500 and 1800 nm respectively(P<0.05).These results show different pathologic liver tissues have different optical properties.It provides a better understanding of the relationship between optical parameters and physiological characteristics in human liver tissues.And it would be very useful for developing a non-invasive,real-time,simple and efficient way for medical management of HCC in the future.     

Effect of Laser in situ Keratomileusis on Accommodation

The accommodative function before and after laser in situ keratomileusis （LASIK） was observed, and the effect of LASIK on accommodation was investigated. In a prospective clinical trial, 48 myopic patients （96 eyes） subject to bilateral LASIK in Refractive Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology （China） from March 2006 to June 2006 were selected and studied. Refractions, accommodative range, amplitude of accommodative response and high frequency component （HFC） of accommodative microfluctuations were measured with NEDIK-730A before and one week and 30 days after operation. Dominant and non-dominant eyes were determined by hole-in-card method. It was found that all of the operative eyes showed an uncorrected visual acuity of 0.8 or better one week postoperatively, and 1.0 or better 30 days postoperatively. Compared with those preoperatively, accommodative range and HFC had no significant difference at first week and 30th day after operation in both dominant eyes and non-dominant eyes （P〉0.05）, but there was a significant difference in the amplitude of accommodative response/accommodative stimulus ratio （A/S） after operation （P〈0.01）, and no significant difference was found in accommodation between one week and 30 days postoperation. No ocular dominance＇s change was noted. There was no significant difference in accommodative function between dominant eyes and non-dominant eyes. It was suggested that LASIK produced no significant effect on accommodation.