PREDOMINANT EXPRESSION OF HUMAN Aγ-IN CONTRAST WITH β-GLOBIN GENE IN MEL CELLS TRANSFECTED WITH THE CONSTRUCT μLCRAγψβδ

A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and adult globins.Similar patterns of human globin gene expression were displayed in the two MEL cell lines transfected with the construct.Inducible expression of the Aγ and β gene was observed during induced cell differentiation.However,the expression level of the Aγ globin gene is much higher than that of the β globin gene in either uninduced or induced MEL transformants.No γ to β switching happened in the stable MEL transformants following a continuous culture.The much more effective enhance of the μLCR on the Aγ globin gene than that on the β globin gene is resulted probably from the fact that the distance between the LCR and the β globin gene is much longer than that between the LCR and the Aγ globin gene in the construct,in comparison with other constructs containing HS2 or μLCR linked to both of γ and β globin genes in different order.Two suggestions can be derived from these results:1) A competition between the γ and β globin gene for interaction with the LCR may indeed present,but only an enough long distance difference between the LCR to the γ and to the β gene can effectively influence the competition;2) Unlike transgenic mice,MEL cells are incapable of reconstructing the regulatory information involved in developmental control when it is provided by a fragment of the β globin gene cluster with limited length.

Trans-acting factors from the human fetal liver bindingto the human ε-globin gene silencer

The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp～-177bp) upstream of thisgene. In order to elucidate its role, the nuclear extractfrom the human fetal liver has been prepared and the interactions between trans-acting factors and this silencerelement have been examined. By using DNasel footprinting assay, a major protected region from -278bp to -235bpwithin this silencer element was identified. Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MW ≈ 32, 28, 26 and 22kD) in the nuclear extractisolated from the human fetal liver, which could specifically bind to this region. Our results suggested that thesetrans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at thefetal stage through the interactions with this silencer.

ISOBUTYRAMIDE ACTIVATES TRANSCRIPTION OF HUMAN FETAL γ-AND MURINE EMBRYONIC εy-GLOBIN GENES

Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant construct μLCRAγψβδβand the stable transformants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rabbit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection assay. The hematological toxicity and electrolyte toxicity ofthe drug were tested.Results. An inducible and dose dependent expression of the human γ , β and mouse α globin gene was observed in the transfected MEL cells. The induction of the human γ globin gene is significant stronger than that of the β globin gene. With 2.5～5 mmol/L isobutyramide, the induction of the human γ globin gene is even more effective than that of mouse α globin gene. After a 15 day injection under the doses of 500～900mg·kg-1·d-1, the level of the mouse embryonic εy globin mRNA could be significantly induced up to 3～4 fold of that of uninjected controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected with different doses of isobutyramide at the interval of 24 hours for 2～4 weeks, were generally within the normal range. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+, Na+, Cl-and CO2 were all within normal range and serum ionic osmotic pressure remained stable as well. Conclusion. Our results suggested that isobutyramide is a weak inducer ofcell differentiation, but it can selectively activate transcription of human γ globin gene at a certain degree, and it can act on early stages of erythroid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have no hematological toxicity. Our results have further proved the potential value of isobutyramide in treatment of β-thalassemia and sickle cell disease.

Study on the Therapeutic Effects and Mechanisms of Human Mesenchymal Stem Cell-Derived Exosomes Carrying NGF Gene in Treating Ischemic Stroke in Rats

Objective:To investigate the therapeutic effects and mechanisms of human mesenchymal stem cell-derived exosomes(hMSCs-Exo)carrying the NGF gene in treating ischemic stroke in rats,aiming to provide new insights and treatment methods for ischemic stroke therapy.Methods:After successful construction of the cerebral ischemia model in 40 male SPF-grade SD rats aged 6-8 weeks,the model rats were randomly divided into 4 groups:Sham group,PBS group,hMSCs-Exo group,and NGF-hMSCs-Exo group,with 10 rats in each group.The rat MCAO model was prepared using the classic filament method,and NGF-hMSCs-Exo were injected via the tail vein into the MCAO model rats.The expression of the NGF gene in brain ischemic tissues,neuronal regeneration,and rat neurological function recovery were observed using TTC staining,memory function evaluation,Western blot,qRT-PCR,and other methods.Results:Compared with the Sham group,neurological deficits were significant in the PBS group(P<0.01).Compared with the PBS group,neurological scores improved in the hMSCs-Exo group and NGF-hMSCs-Exo group(P<0.05).Compared with the hMSCs-Exo group,the improvement in neurological deficits was more significant in the NGF-hMSCs-Exo group(P<0.05).The infarct area after NGF-hMSCs-Exo intervention was significantly reduced(P<0.05)compared with the Sham group.Compared with the PBS group,relative expression levels of NGF mRNA and protein decreased,while Caspase-3 mRNA and protein expression significantly increased in the PBS group(P<0.01).Compared with the PBS group and hMSCs-Exo group,there were differences in NGF and Caspase-3 mRNA and protein expression in the NGF-hMSCs-Exo group rat brain tissues(P<0.05).Conclusion:Treatment with human mesenchymal stem cell-derived exosomes carrying the NGF gene improves cognitive function and exerts protective effects on SD rats while inhibiting apoptotic levels in cells.

Studies on DNA-protein interactions in the upstream regulatory region of the human ε-globin gene promoter

The erythroid- and developmental stage-specific expression of the human ε-globin gene is controlled, in part,by the 5’-flanking DNA sequence of this gene. In the present study, we have used DNA-protein binding assays to identify trans-acting factors which regulate the temporal expression of the human ε-globin gene during development. Using gel mobility shift assays and DNasel footprinting assays, a nuclear protein factor (termed ε-SSF1) in the nuclear extracts from mouse haematopoietic tissues at d 11 and d 13 of gestation was identified. It could specifically bind to the positive control region (between -535 and -453bp) of the human ε-globin gene. We speculated that the E-SSF1 might be an erythroid- and developmental stage-specific activator. In addition, we found another nuclear protein factor (termed ε-R1) in the nuclear extract from mouse fetal liver at d 18 of gestation, which could strongly bind to the silencer region (between -392 and -177bp) of this gene. Therefore, we speculated that the ε-R1 might be an erythroid- and developmental stagespecific repressor. Our data suggest that both ε-SSF1 and ε-R1 might play important roles in developmental regulation of the human ε-globin gene expression during the early embryonic life. On the other hand, we observed that the binding patterns of nuclear proteins from three cell lines (K562, HEL and Raji) to these regulatory regions were partially different. These results suggest that different trans-acting factors in K562, HEL and Raji cells might be responsible for activating or silencing the human ε-globin gene in three different cell lines.

A DNA-binding protein factor in K562 nuclear extract interacts with positive control region (PCR) in the 5'flanking sequence of human β-globin gene

It has been known that there are at least three regulatory regions (NCR1, NCR2 and PCR) in the 5'-flanking sequence (from -610 bp to +1bp) of human β-globin gene and that the function of PCR is unique to the human erythroleukemia (K562) cells. Here we have detected a DNA-binding protein factor (termed NFEa) in K562 cells, which can bind specifically to the PCR of human β-globin gene. The sequence of the binding site is 5'ACTGATG3' (between -222 bp and -216 bp). The NFEa is erythroid-specific and perhaps specific for K562 cells. It seemed that this factor differed from the erythroid-specific tran-scriptional factor (NFE-1) using competition assay. The presence of the NFEa further supported that the function of the cis-acting element PCR was specific for K562 cells, and helps us to understand the mechanism of the regulation of the expression of human β-globin gene in the human K562 cells.

Proteins binding to the 5'-flanking regulatory elements of the human β-globin gene

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human β-globin gene, two negative control regions(NCR1,-610 to -490 bp;NCR2, -338 to-233bp), was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in part for the silence of β-globin gene in the embryonic and fetal stages.

The 5'-flanking cis-acting elements of the human ε-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.

Identification of a NF-кB site in the negative regulatory element (ε-NRAII) of human ε-globin gene and its binding protein NF-кB p50 in the nuclei of K562 cells

The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5’ flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-кB consensus sequence resides in the negative regulatory element (-3028bp～ -2902bp, termed ε-NRAII) 5’ to the cap site of this gene. NRF DNA fragment (-3010bp～ -2986bp) containing the NF-кB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-кB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-кB motif) could not, suggesting that the binding protein is a member of NF-кB/Rel family. Western blot assay demonstrated that the molecular weight of NF-кB protein in the nuclei of K562 cells is 50ku. We suggested that NF-кB p50 may play an important role in the regulation of human c-globin gene expression.

Identification of a NF-кB site in the negative regulatory element (ε-NRAII) of human ε-globin gene and its binding protein NF-кB p50 in the nuclei of K562 cells

The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5' flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-кB consensus sequence resides in the negative regulatory element (-3028bp～ -2902bp, termed ε-NRAII) 5' to the cap site of this gene. NRF DNA fragment (-3010bp～ -2986bp) containing the NF-кB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-кB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-кB motif) could not, suggesting that the binding protein is a member of NF-кB/Rel family. Western blot assay demonstrated that the molecular weight of NF-кB protein in the nuclei of K562 cells is 50ku. We suggested that NF-кB p50 may play an important role in the regulation of human c-globin gene expression.

Transfection of the Human Sodium/Iodide Symporter(NIS) Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.

人类“绿色基因”假说(Human"Green-Gene"Hypothesis):核心内容、科学佐证与实践意义

首先,对人类“绿色基因”的形成进行探讨,包括人类(动物)与植物共同维持大气中碳氧平衡、人类(动物)与植物互为食物链关系、人体功能中存在植物性功能、绿色是人类眼睛最易看到的颜色、人类血红蛋白的结构相似于叶绿素结构、作为人体呼吸器官的肺部相似于树木地上部形状(树形)、人类肠道绒毛相似于植物根系毛细根等方面。进而对支持早期人类生活的植物类型与栽培植物诞生进行研究。在此基础上,提出人类“绿色基因”假说及其核心内容,归纳该假说的科学佐证。人类“绿色基因”假说从整体的、发展的思维解释人类与植物关系,奠定了园艺疗法、园林康养、森林康养的坚实基础,并指出接触植物、进行园艺操作与田园劳作是人们实现接地、激发触觉潜在功能的途径。

Cloning and expression of MXR7 gene in human HCC tissue

AIM To clone and identify the whole cDNA ofMXR7 gene and to find out its expression inhuman HCC,and normal tissues.METHODS The DNA primers were designed andsynthesized according to the whole cDNAsequence of MXR? gene.The cDNA of humanHCC was taken as the template while the cDNA ofMXR7 gene was synthesized by polymerasechain reaction（PCR）.Recombinant DNAconforming to reading frame was constructed byconnecting purified PCR product of the cDNA ofMXR? gene with expression vector pGEX-5X-1 offusion protein.The plasmid MXRT/pGEX-5X-1was identified by sequencing.Using 32p labeledMXR? cDNA as probe,MXR7 mRNA expressionwas detected by Northern blot analysis in 12different human normal tissues,7 preoperativelyuntreated non-liver tumor tissues,30preoperatively untreated HCC,theparacancerous liver tissues and 12 normal livertissues samples.RESULTS Restriction enzyme and sequenceanalysis confirmed that the insertion sequence invector pGEX-5X-1 was the same as the cDNAsequence of MXR7 gene.Northern blot analysisshowed no expression of MXR? mRNA in 12 kindsof normal human tissues including liver,7 tumortissues in other sites and 12 normal livertissues,the frequencies of MXR7 mRNA expression in HCC and paracancerous livertissues were 76.6% and 13.3%,respectively.The frequency of MXR7 mRNA expression in HCCwithout elevation of serum AFP and in HCC【5cm was 90%（9/10）and 83.3%（5/6）,respectively.CONCLUSION MXR7 mRNA is highly expressedin human HCC,which is specific and occurs at anearly stage of HCC,suggesting MXR7 mRNA canbe a tumor biomarker for HCC.The detection ofMXR7 mRNA expression in the biopsied livertissue is helpful in discovering early subclinicalliver cancer in those with negative serum AFP.

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Identification and characterization of the BGR-like gene with a potential role in human testicular development/spermatogenesis

Aim: To investigate the roles of the BGR-like gene in testicular development/spermatogenesis. Methods: A human testis cDNA microarray was hybridized with probes from human adult testes and embryo testes. The differentially expressed clones were sequenced and analyzed. Expression of the BGR-like gene was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Results: A new gene exhibiting 50-fold difference in expression level between adult and fetal human testes was cloned and named the BGR-like gene. The cDNA consisted of 2500 nucleotides and had an open reading frame of 1437 nucleotides encoding a putative protein of 497 amino acid residues. Homologous comparison showed that the BGR-like gene was a new alternative splicing variant of the BGR gene and had sequence homology with the bubblegum gene of human, mouse, rat and Drosophilia. Protein motif analysis of the BGR-like gene revealed that it contained a conserved adenosine monophosphate (AMP)-binding domain and a fatty acyl-CoA synthetase signature motif which existed in all acyl-CoA synthetases. The BGR-like gene transcript was imperceptibly expressed in human fetal testes, highly in human adult testes and moderately in elderly testes and human Leydig cells. RT-PCR-based tissue distribution experiments showed that the BGR-like gene was exclusively expressed in testes and was a testes-specific isoform of the BGR gene. A BGR-like gene transcript was not detected in some azoospermic testes. Conclusion: The BGR-like gene may play an important role in spermatogenesis/testicular development and may be correlated with male infertility.

Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and historic acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methyla-tion-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

APC and K-ras gene mutation in aberrant crypt foci of human colon

AIM:To study the genetic alteration in ACF and to define the possibility that ACF may be a very early morphological lesion with molecular changes,and to explore the relationship between ACF and colorectal adenoma even carcinoma. METHODS: DNA from 35 CRC, 15 adenomas, 34 ACF and 10 normal mucus was isolated by means of microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well as the mutation cluster region (MCR) of APC gene was performed. RESULTS: K-ras gene mutation frequency in ACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/15), and 14.3% (5/35) respectively, showing no difference (P 】 0.05) in K-ras gene mutation among three pathologic procedures. The K-ras gene mutation in adenoma, carcinoma and 4 ACF restricted in codon 12 (GGT GAT), but the other 2 mutations from ACF located in codon 13 (GGC GAC). K-ras gene mutation was found more frequently in older patients and patients with polypoid cancer. No mutation in codon 61 was found in the three tissue types. Mutation rate of APC gene in adenoma and carcinoma was 22.9% (8/35) and 26.7% (4/15), which was higher than ACF (2.9%) (P 【0.05). APC gene mutation in carcinoma was not correlated with age of patients, location, size and differentiation of tumor. CONCLUSION: ACF might be a very early morphological lesion in the tumorogenesis of colorectal tumor. The morphological feature and gene mutation status was different in ACF and adenoma. ACF is possibly putative microadenoma that might be the precursor of adenoma. In addition, the development of a subgroup of colorectal carcinomas might undergo a way of normal epithelium ACF carcinomas .

Upreguiation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved

Identification and Validation of Candidate Radiation-responsive Genes for Human Biodosimetry

The aim of the present study is to analyze the global research trend of radiation-responsive genes and identify the highly reproducible radiation-responsive genes. Bibliometric methods were applied to analyze the global research trend of radiation-responsive genes. We found 79 publications on radiation-responsive genes from 2000 to 2017. A total of 35 highly reproducible radiation-responsive genes were identified. Most genes are involved in response to DNA damage, cell proliferation, cell cycle regulation, and DNA repair.The p53 signal pathway was the top enriched pathway. The expression levels of 18 genes in human B lymphoblastoid cell line（AHH-1） cells were significantly up-regulated in a dose-dependent manner at 24 h after exposure to 0-5 Gy ^60 Coγ-ray irradiation. Our results indicate that developing a gene expression panel with the 35 high reproducibility radiation-responsive genes may be necessary for qualitative and quantitative assessment after exposure.

Enhancement of mouse germ cell-associated genes expression by injection of human umbilical cord mesenchymal stem cells into the testis of chemical-induced azoospermic mice

Various methods are currently under investigation to preserve fertility in males treated with high-dose chemotherapy and radiation for malignant and nonmalignant disorders. Human umbilical cord mesenchymal stem cells （HUC-MSCs）, which possess potent immunosuppressive function and secrete various cytokines and growth factors, have the potential clinical applications. As a potential alternative, we investigate whether injection of HUC-MSCs into the interstitial compartment of the testes to promote spermatogenic regeneration efficiently. HUC-MSCs were isolated from different sources of umbilical cords and injected into the interstitial space of one testis from 10 busulfan-treated mice （saline and HEK293 cells injections were performed in a separate set of mice） and the other testis remained uninjected. Three weeks after MSCs injection, Relative quantitative reverse transcription polymerase chain reaction was used to identify the expression of 10 of germ cell associated, which are all related to meiosis, demonstrated higher levels of spermatogenic gene expression （2-8 fold） in HUC-MSCs injected testes compared to the contralateral uninjected testes （five mice）. Protein levels for germ cell-specific genes, miwi, vasa and synaptonemal complex protein （Scp3） were also higher in MSC-treated testes compared to injected controls 3 weeks after treatment. However, no different expression was detected in saline water and HEK293 cells injection control group. We have demonstrated HUC-MSCs could affect mouse germ cell-specific genes expression. The results also provide a possibility that the transplanted HUC-MSCs may promote the recovery of spermatogenesis. This study provides further evidence for preclinical therapeutic effects of HUC-MSCs, and explores a new approach to the treatment of azoospermia.