共查询到20条相似文献,搜索用时 15 毫秒
1.
Yan Jun HUAN Jiang ZHU Bing Teng XIE Jian Yu WANG Shi Chao LIU Yang ZHOU Qing Ran KONG Hong Bin HE Zhong Hua LIU 《The Journal of reproduction and development》2013,59(5):442-449
The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low.
In most cloned embryos, epigenetic reprogramming is incomplete, and usually the
genome is hypermethylated. The DNA methylation inhibitor 5-aza-2’-deoxycytidine
(5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT
embryos in previous studies. However, the parameters of 5-aza-dC treatment among
species are different, and whether 5-aza-dC could enhance the developmental
competence of porcine cloned embryos has still not been well studied. Therefore, in
this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor
nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with
5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine
cloned embryos were investigated by assessing pseudo-pronucleus formation,
developmental potential and pluripotent gene expression of these reconstructed
embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation
level in PFF (0 nM vs. 10 nM vs. 25 nM
vs. 50 nM, 58.70% vs. 37.37%
vs. 45.43% vs. 39.53%, P<0.05), but did not
improve the blastocyst rate of cloned embryos derived from these cells. Treating
cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst
rate compared with that of the untreated group. Furthermore, treating cloned embryos,
but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post
activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for
control, respectively, P<0.05) and enhanced the expression levels of pluripotent
genes (Oct4, Nanog and Sox2) up to
those of in vitro fertilized embryos during embryo development. In
conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the
developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus
formation and improvement of pluripotent gene expression. 相似文献
2.
Kanokwan SRIRATTANA Mariena KETUDAT-CAIRNS Takashi NAGAI Masahiro KANEDA Rangsun PARNPAI 《The Journal of reproduction and development》2014,60(5):336-341
Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several
species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned
embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp
buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results
showed that there was no significant difference in the rates of fusion (82–85%), cleavage (79–84%) and development to the
8-cell stage (59–65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos
were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the
satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite
DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than
those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the
methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the
in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA
methylation level was observed. 相似文献
3.
DNA methylation and expression of imprinted genes are associated with the viability of different sexual cloned buffaloes 下载免费PDF全文
Z Ruan X Zhao X Qin C Luo X Liu Y Deng P Zhu Z Li B Huang D Shi F Lu 《Reproduction in domestic animals》2018,53(1):203-212
The DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming of donor cells in somatic cell nuclear transfer (SCNT). However, the effects of sexual distinction on the DNA methylation of imprinted genes in cloned animals have seldom been reported. In this study, we analysed the DNA methylation status of three imprinted genes (Xist, IGF2 and H19) from liveborn cloned buffaloes (L group, three female and three male), stillborn cloned buffaloes (S group, three female and three male) and natural reproduction buffaloes (N group, three female and three male), using bisulphite sequencing polymerase chain reaction (BS‐PCR). The expression levels of these imprinted genes were also investigated by quantitative real‐time PCR (QRT‐PCR). The DNA methylation levels of H19 were not significantly different among the groups. However, the Xist in female and IGF2 in male of the S group were found to be significantly hypomethylated in comparison with the same sexual buffaloes in L group and N group (p < .05). Furthermore, the expression levels of Xist, IGF2 and H19 in the stillborn female cloned buffaloes of S group were significantly higher than that of the female buffaloes in the L group and N group (p < .05). The expression levels of IGF2 and H19 in the stillborn male cloned buffaloes in the S group were significantly higher than that of the male buffaloes in the L group and N group (p < .05). These results indicate that Xist may be associated with the viability of female cloned buffaloes, and IGF2 may also be related to the viability of male cloned buffaloes. 相似文献
4.
Lorthongpanich C Laowtammathron C Chan AW Ketudat-Cairns M Parnpai R 《The Journal of reproduction and development》2008,54(5):306-313
This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos. 相似文献
5.
6.
Nozomi TAKAHASHI Eito YAMAGUCHI Yukiko KAWABATA Tomohiro KONO 《The Journal of reproduction and development》2015,61(1):7-12
The distal region of mouse chromosome 12 harbors the Dlk1–Dio3 domain, is essential for normal development and encodes maternally expressed noncoding RNAs (ncRNAs), including Gtl2 as well as paternally expressed proteins.Gtl2 works as a tumor suppressor in several types of human cancer cell lines; however, whether this reflects its function in vivo is unknown. Deleting Gtl2 from the maternal allele (Gtl2(–/+)) results in loss of expression of Gtl2 and decreased expression of downstream ncRNAs, including many miRNAs. To determine the role of ncRNAs in tumorigenesis, we induced teratomas by engrafting E6.5 embryos (wildtype or Gtl2(–/+)) under the kidney capsule of scid mice. Some teratomas derived from the Gtl2(–/+) embryos exhibited hypertrophic growth, suggesting that
ncRNAs, including Gtl2, may act as tumor suppressors in vivo. Microarray analysis of miRNAs expressed by Gtl2(–/+) teratomas revealed decreased expression of 28 miRNAs encoded by the Dlk1–Dio3 domain, low expression of embryonic stem cell-specific miRNAs and dysregulation of miRNAs involved in tumorigenesis. This study suggests that downregulation of ncRNAs in the Dlk1-Dio3 domain leads to enhanced teratoma growth and repression of stem cell markers. 相似文献
7.
Involvement of histone H2B monoubiquitination in the regulation of mouse preimplantation development
Masatoshi OOGA Masataka G. SUZUKI Fugaku AOKI 《The Journal of reproduction and development》2015,61(3):179-184
Histone H2B monoubiquitination (H2Bub1) plays an important role in developmental regulation in various vertebrate species. However, the role of H2Bub1 in mammalian preimplantation development remains unclear. In the present study, we examined the role of H2Bub1 in the regulation of mouse preimplantation development. Based on immunocytochemical analysis using an anti-H2Bub1 antibody, no H2Bub1 signal was detected in the metaphase chromosomes of unfertilized oocytes or the pronuclei of early 1-cell stage embryos, but a weak signal was observed in late 1-cell stage embryos. The signal increased after cleavage into the 2-cell stage, and thereafter a strong signal was observed until the blastocyst stage. To assess the significance of H2Bub1 in the regulation of preimplantation development, RNF20 (an H2B-specific ubiquitin E3 ligase) was knocked down using small interfering RNA (siRNAs). In embryos treated with siRNA, the levels of Rnf20 mRNA and H2Bub1 decreased
at the 4-cell and morula stages. Although these embryos developed normally until the morula stage, only one-third developed into the blastocyst stage. These results suggested that H2Bub1 is involved in the regulation of preimplantation development. 相似文献
8.
Primordial germ cells (PGCs) from day 27 porcine fetuses have often been isolated to establish pluripotent embryonic germ (EG) cell lines, but little is known regarding their imprinted gene status. In our study, we attempted to detect the imprinted gene expression of cloned embryos and EG cells derived from individual PGC of day 27 and day 35, using single nucleotide polymorphism (SNP) analysis of the paternally expression gene 10 (PEG10) as a sign of parental‐origin‐specific expression. The results showed biallelic gene expression of the SNP that occurred in EG cell colonies and almost all of the cloned blastocysts, demonstrating that aberrant imprinted gene expression of PEG10 occurs in the day 27 porcine PGCs, whereas monoallelic expression of the PEG10 gene occurs in all the PGC clones derived from day 35 PGCs. In addition, the same imprinted gene status was observed for blastocysts derived from both male and female PGCs, indicating that the parental genomic imprinting is erased in male and female germlines. 相似文献
9.
Rui Kano Takayuki Yano Kota Nagamatsu Haruhiko Maruyama Hiroshi Kamata Atsuhiko Hasegawa 《Research in veterinary science》2009,87(1):64-66
The effect of down-regulation of Mcl-1 expression by small interfering RNA (siRNA) against the canine Mcl-1 gene on apoptosis was investigated by transfecting CF33 (canine mammary gland tumor cell line) with siRNA using cationic liposomes. The siRNA against canine Mcl-1 increased the rate of apoptotic cells and decreased the numbers of viable cells. Further, sequence-specific down-regulation of Mcl-1 expression was measured by real time-PCR and Western blot analysis. The siRNA directed against the Mcl-1 gene reduced both the mRNA and protein expression in the CF33. Our study suggests the importance of Mcl-1 in canine mammary tumors for inducing apoptosis and reinforces using Mcl-1 as a putative therapeutic target in canine mammary gland tumor. 相似文献
10.
Seon-Hyang KIM Ming-Hui ZHAO Shuang LIANG Xiang-Shun CUI Nam-Hyung KIM 《The Journal of reproduction and development》2015,61(4):261-268
Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05)
enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria. 相似文献
11.
Although the technique of interspecies somatic cell nuclear transfer can be used to increase the population size of endangered mammals, the mitochondrial heteroplasmy in cloned embryos and animals makes this idea doubtful. In present study, goat–sheep cloned embryos were constructed by fusing goat foetal fibroblasts (GFFs) into sheep oocytes and then cultured in vitro to investigate the capability of sheep oocyte dedifferentiating GFF nucleus. Moreover, at each stage of 1‐ (immediately after fused), 2‐, 4‐, 8‐, 16‐cell, morula and blastocyst, the copy number of mtDNA from GFF and sheep oocyte was examined using real‐time PCR. The results showed that: 7.4% of the fused cloned embryos can develop to the blastocyst stage; in the process of one cell to the morula stage, the copy number of two kinds of mtDNA was stable relatively; however, in the process of morula to the blastocyst stage, the decreasing in the copy number of GFF‐derived mtDNA, while the increasing in sheep oocyte‐derived, resulted in their ratio of decreasing sharply from 2.0 ± 1.0% to 0.012 ± 0.004%. This study demonstrates that: (i) the goat–sheep cloned embryos have the ability to develop to blastocyst in vitro; (ii) from the morula stage to the blastocyst stage of goat–sheep cloned embryos, goat derived mitochondria can be gradually replaced with those from sheep oocyte. 相似文献
12.
Yuria UMEMURA Ryosuke MIYAMOTO Rie HASHIMOTO Kyoko KINOSHITA Takuya OMOTEHARA Daichi NAGAHARA Tetsushi HIRANO Naoto KUBOTA Kiichi MINAMI Shogo YANAI Natsumi MASUDA Hideto YUASA Youhei MANTANI Eiko MATSUO Toshifumi YOKOYAMA Hiroshi KITAGAWA Nobuhiko HOSHI 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2015,77(12):1587-1598
Mammalian sexual fate is determined by the presence or absence of sex determining
region of the Y chromosome (Sry) in the “bipotential” gonads.
Recent studies have demonstrated that both male and female sexual development are induced
by distinct and active genetic pathways. Breeding the Y chromosome from Mus m.
domesticus poschiavinus (POS) strains into C57BL/6J (B6J) mice
(B6J-XYPOS) has been shown to induce sex reversal (75%: bilateral ovary, 25%:
true hermaphrodites). However, our B6N-XYPOS mice, which were generated by
backcrossing of B6J-XYPOS on an inbred B6N-XX, develop as males (36%: bilateral
testis with fertility as well as bilateral ovary (34%), and the remainder develop as true
hermaphrodites. Here, we investigated in detail the expressions of essential sex-related
genes and histological features in B6N-XYPOS mice from the fetal period to
adulthood. The onsets of both Sry and SRY-box 9 (Sox9) expressions as determined
spatiotemporally by whole-mount immunohistochemistry in the B6N-XYPOS gonads
occurred 2–3 tail somites later than those in B6N-XYB6 gonads, but earlier than
those in B6J-XYPOS, respectively. It is possible that such a small difference
in timing of the Sry expression underlies testicular development in our
B6N-XYPOS. Our study is the first to histologically show the expression and
ectopic localization of a female-related gene in the XYPOS testes and a
male-related gene in the XYPOS ovaries. The results from these and previous
experiments indicate that the interplay between genome variants, epigenetics and
developmental gene regulation is crucial for testis development. 相似文献
13.
Jae Yeon HWANG Kwang-Hwan CHOI Dong-Kyung LEE Seung-Hun KIM Eun Bae KIM Sang-Hwan HYUN Chang-Kyu LEE 《The Journal of reproduction and development》2015,61(6):533-540
X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between
male and female eutherians. This process is observed in early eutherian embryo development in a
species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the
process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent
insights into the process and its regulation have been restricted in mouse species despite the evolutionary
diversity of the process and molecular mechanism among the species. OCT4A is one of the
represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST
repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in
porcine preimplantation embryos. Three X-linked genes, XIST,
LOC102165544, and RLIM, were selected in present study because their
orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were
positively correlated with XIST and LOC102165544 in female blastocysts.
Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated
blastocysts with increased XIST expression levels. However, increased XIST
expression was not observed when exogenous OCT4A was obtained from early blastocysts. These
results suggest the possibility that OCT4A would be directly or indirectly involved in
XIST expression in earlier stage porcine embryos rather than blastocysts. 相似文献
14.
Jiang BIAN Tao LI Chenhui DING Weijie XIN Bo ZHU Canquan ZHOU 《The Journal of reproduction and development》2013,59(3):288-295
To completely avoid ice crystal formation and thus get a higher survival rate,
vitrification methods have been commonly used for cryopreservation of oocytes and embryos.
However, currently used vitrification methods for oocytes and embryos are not suitable for
the cryopreservation of preantral follicles (PFs). In the present study, stainless steel
mesh was fabricated into mini mesh cups to vitrify isolated PFs. Moreover, isolated
follicles were encapsulated and then subjected to vitreous cryopreservation to facilitate
in vitro culture/maturation of follicles after warming. The results
showed that the percentages of viable follicles did not differ significantly between the
vitrification group and fresh group soon after warming (81.25% vs.
85.29%, P>0.05) and after a 7-day culture period (77.78% vs. 83.33%,
P>0.05). No difference in mean follicular diameter was observed between cryopreserved
and fresh follicles when cultured in vitro. Transmission electron
microscopic analysis revealed that vitreous cryopreservation could maintain the
ultrastructure of follicles in alginate beads. In conclusion, the present vitrification
method could efficiently cryopreserve isolated human ovarian follicles encapsulated by
calcium alginate, which could be put into immediate use (in vitro
culture/ maturation) after warming. However, more follicles and some detailed biochemical
analyses are required to further investigate the effects of vitrification on the long-term
growth of human encapsulated PFs. 相似文献
15.
S. Unterer K. Busch M. Leipig W. Hermanns G. Wolf R.K. Straubinger R.S. Mueller K. Hartmann 《Journal of veterinary internal medicine / American College of Veterinary Internal Medicine》2014,28(1):52-58
Background
Etiology of hemorrhagic gastroenteritis (HGE) syndrome in dogs is unknown and histopathologic and microbial investigations have only been performed post mortem.Objective
To identify characteristic intra vitam endoscopic and histologic mucosal lesions, as well as bacterial species, within the mucosa of dogs with HGE.Animals
Ten dogs diagnosed with HGE were included. Eleven dogs with gastroduodenoscopy and different intestinal diseases were used as controls for microbial changes. Dogs pretreated with antibiotics or diagnosed with any disease known to cause bloody diarrhea were excluded from the study.Methods
In this prospective study, gastrointestinal biopsies were collected from 10 dogs with HGE. Endoscopic and histologic changes were assessed according to WSAVA guidelines. Biopsies from the stomach, duodenum, ileum, and colon were investigated by histology and by immunohistochemistry for the presence of Clostridium spp. and parvovirus. The first duodenal biopsy taken with a sterile forceps was submitted for bacterial culture.Results
Acute mucosal lesions were only found in the intestines, not in the stomach. Clostridium spp., identified as Clostridium perfringens in 6/9 cases, were detected on the small intestinal mucosa in all dogs with HGE, either by culture or immunohistopathology. In the control group, C. perfringens could only be cultured in one of 11 dogs.Conclusions and Clinical Importance
The results of this study demonstrate an apparent association between C. perfringens and the occurrence of acute hemorrhagic diarrhea. The term “HGE,” which implies the involvement of the stomach, should be renamed as “acute hemorrhagic diarrhea syndrome.” 相似文献16.
17.
Chommanart THONGKITTIDILOK Theerawat THARASANIT Nucharin SONGSASEN Thanida SANANMUANG Sirirak BUARPUNG Mongkol TECHAKUMPHU 《The Journal of reproduction and development》2015,61(4):269-276
This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group
or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages. 相似文献
18.
Jin-A LEE Bock-Gie JUNG Tae-Hoon KIM Yun-Mi KIM Hong-Bum KOH Bong-Joo LEE 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2015,77(9):1087-1094
Biotite and bentonite are phyllosilicate minerals that were originally used in
industrial applications. Several beneficial activities of them have recently been
reported, especially regulation of the immune system and antimicrobial effects. Therefore,
we investigated the immune-enhancing and bacterial clearance effects of a biotite and
bentonite mixture (BBM) on experimental infection of Salmonella enterica
serovar Typhimurium (S. Typhimurium) to determine whether the BBM could
be used as an alternative antibiotic. We administered 1% or 2% BBM as a feed supplement.
We then evaluated the bacterial clearance effects of the BBM against S.
Typhimurium. We also evaluated the immune-enhancing effect of the BBM through several
immunological experiments that included examination of the lysozyme activity,
CD4+/CD8+ T lymphocyte ratio and the T-helper type 1 (Th 1)
cytokine profile. The clinical signs of S. Typhimurium and the number of
viable bacteria in feces and tissues were significantly decreased in both BBM groups,
especially in the 2% BBM group. The BBM also markedly enhanced the lysozyme activity,
CD4+/CD8+ T lymphocyte ratio and expression levels of IFN-γ and
IL-12 in S. Typhimurium-challenged pigs. Therefore, the BBM could be a
good candidate as an alternative antibiotic that improves Th 1-specific immune responses
and the bacterial clearance effect. 相似文献
19.
Wei Cong Hong Jin Chengda Jiang Weiyao Yan Mingqiu Liu Jiulian Chen Xiaoping Zuo Zhaoxin Zheng 《Veterinary research》2010,41(3)
In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 109 CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID50 of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 109 CFU of p3D-NT56/S. cho were protected in 9 days. 相似文献
20.
Adhesion through microbial surface components that recognize adhesive matrix molecules is an essential step in infection for most pathogenic bacteria. In this study, we report that LigB interacts with fibronectin (Fn) through its variable region. A possible role for LigB in bacterial attachment to host cells during the course of infection is supported by the following observations: (i) binding of the variable region of LigB to Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner reduces the adhesion of Leptospira, (ii) inhibition of leptospiral attachment to Fn by the variable region of LigB, and (iii) decrease in binding of the variable region of LigB to the MDCK cells in the presence of Fn. Furthermore, we found a significant reduction in binding of the variable region of LigB to Fn using small interfering RNA (siRNA). Finally, the isothermal titration calorimetric results confirmed the interaction between the variable region of LigB and Fn. This is the first report to demonstrate that LigB binds to MDCK cells. In addition, the reduction of Fn expression in the MDCK cells, by siRNA, reduced the binding of LigB. Taken together, the data from the present study showed that LigB is a Fn-binding protein of pathogenic Leptospira spp. and may play a pivotal role in Leptospira-host interaction during the initial stage of infection. 相似文献