唾液腺腺样囊性癌XT-Ⅰ与XT-Ⅱ基因共沉默的实验研究
发布时间：2018-08-14 10:03
【摘要】：目的: 唾液腺腺样囊性癌（salivary adenoid cystic carcinoma, SACC）是最常见的唾液腺恶性肿瘤之一，侵袭性强，可转移，易复发，预后不佳，迄今尚无较理想的治疗措施。 SACC中的肿瘤性肌上皮细胞（neoplastic myoepithelial cells, NMCs）可以分泌蛋白多糖（proteoglycans, PGs），构成肿瘤的细胞外基质（extracellular matrix, ECM）。在SACC中，过多的ECM逐渐堆积，形成囊腔样结构，从而呈现出SACC特有的组织学特点。富含PGs的ECM，构成了SACC细胞的大分子微环境（macromolecules microenvironment）。SACC细胞在这一微环境内生存，继而进展，出现增殖、分化、侵袭、转移、复发等一系列生物学行为。目前认为，PGs是SACC的形态学表现和生物学行为必不可少的物质基础，参与肿瘤的发生和发展过程。 人木糖基转移酶（xylosyltransferase, XT）是人体PGs生物合成的起始酶和限速酶，催化此合成过程的效率—限制阶段（rate-limited step），它的活性真实反映PGs的生物合成率。人XT有两个亚型（isoform）：XT-I和XT-II，催化作用几乎相同，但是在人体不同细胞的表达有所差异。 RNA干扰（RNA interference, RNAi）是目前最有效的基因沉默（genesilencing）技术，可以特异、高效地阻断特定基因的表达。腺病毒载体是将外源基因导入细胞内的常用媒介，同时表达一个以上目的基因的腺病毒载体，可同时发挥多个基因的功能，提高了腺病毒载体的利用率。 本研究拟构建一个同时沉默XT-I和XT-II基因的质粒载体，即编码2条shRNA的干扰质粒，腺病毒包装，获得重组腺病毒rAd5-shRNA-WJ7＋WJ4；通过感染SACC-83细胞，干扰XT-I和XT-II的表达，阻抑PGs合成，检测其沉默效果；建立SACC-83的裸鼠移植瘤模型，瘤体内注射重组腺病毒，观察其对移植瘤凋亡的影响。旨在探讨SACC中XT-I和XT-II对PGs的调控作用，以及XT-I和XT-II共沉默对细胞凋亡的影响，并为唾液腺肌上皮性肿瘤的生物治疗提供更多的依据。 方法： 1构建分别靶向抑制人XT-I基因和人XT-II基因的两个重组质粒 设计靶向抑制人XT-I基因的shRNA-WJ4和靶向抑制人XT-II基因的shRNA-WJ7的干扰位点和引物序列，合成shRNA-WJ4和shRNA-WJ7的单链目的基因片段，分别与质粒pGenesil-1.2和pGenesil-1.1的线性化载体连接，扩增质粒，酶切鉴定，确定获得重组质粒pGenesil-1.2-shRNA-WJ4和pGenesil-1.1-shRNA-WJ7。 2构建同时靶向抑制人XT-I基因和人XT-II基因的共沉默质粒 双酶切以上两个重组质粒，回收大小片段，WJ7大片段与WJ4小片段的连接，扩增质粒，酶切鉴定，测序验证共沉默质粒shRNA-WJ7＋WJ4。 3重组腺病毒质粒的制备 从质粒pGenesil上通过LR体外同源重组将shRNA-WJ7＋WJ4表达框转移至pGSadeno腺病毒表达载体上，扩增质粒，酶切鉴定。 4包装共沉默重组腺病毒 制备线性化腺病毒DNA，连接重组腺病毒质粒片段，转染HEK293细胞，放大培养共沉默重组腺病毒rAd5-shRNA-WJ7＋WJ4。 5构建阴性对照重组腺病毒rAd5-shRNA-HK 6重组腺病毒感染细胞 培养SACC-83细胞，分3组：SACC-83-WJ7＋WJ4组（沉默组）、SACC-83-HK组（空载体组）、SACC-83组（未感染组），确定最佳MOI值为100，细胞感染48h的荧光最强，计算感染率。 7检测感染48h后3组细胞XT-I和XT-I mRNA的表达 TRIzol法提取细胞总RNA，测定RNA纯度、浓度、完整性，逆转录合成cDNA第一链，实时荧光定量PCR（Real-Time PCR）检测3组细胞XT-I和XT-I mRNA的相对表达量，计算沉默率。 8检测感染48h后3组细胞培养液中PGs的含量 用标准品建立浓度-吸光度标准曲线，生物染色法测定PGs含量，计算抑制率。 9裸鼠移植瘤模型的建立及分组 4周龄雌性BALB/c-nu裸小鼠32只，14～15g，无特定病原体（specificpathogen-free, SPF）条件饲养，每只于左侧背部接种SACC-83细胞2.5×106个／250μl；62d后，选取瘤体最长径在1cm以上的24只，随机数字表法分为3组：SACC-83-WJ7＋WJ4组（沉默组）、SACC-83-HK组（空载体组）、SACC-83组（未感染组），每组8只。 10重组腺病毒裸鼠移植瘤内注射 接种肿瘤细胞后62d，首次瘤内注射重组腺病毒，SACC-83-WJ7＋WJ4组（沉默组）注射共沉默腺病毒rAd5-shRNA-WJ7＋WJ4，SACC-83-HK组（空载体组）注射阴性对照腺病毒rAd5-shRNA-HK，SACC-83组（未感染组）注射PBS，每只4×109PFU／200μl，每周注射一次，共5次。 11标本采集 3组裸鼠首次注射腺病毒5周后断颈处死，分离移植瘤，测量体积，新鲜瘤组织立即分切为4份，1份80C冻存，3份用于以下实验。 12流式细胞术样品制备和凋亡检测 70%乙醇固定标本，剪碎法＋网搓法制备单细胞悬液，PI染色，流式细胞术检测3组样品的细胞凋亡情况。 13HE染色样品制备和观察 10%福尔马林固定标本、石蜡包埋、切片、HE染色、光学显微镜观察3组标本的形态学变化。 14透射电镜超薄切片样品制备和观察 标本分切至小于1mm3，4%戊二醛前固定、1%锇酸后固定、环氧树脂包埋、超薄切片、醋酸铀和柠檬酸铅双重染色30min、透射电镜观察细胞凋亡情况。 结果： 1分别靶向XT-I、XT-II和阴性对照的siRNA序列设计结果shRNA-WJ4CAGGCAGCCCATCAAACCTshRNA-WJ7CGTCTCCTCAAGGCCGTTTATshRNA-HK GACTTCATAAGGCGCATGC 2酶切和琼脂糖凝胶电泳鉴定结果、测序验证结果均证明质粒正确。 3重组腺病毒感染SACC-83细胞的效率 重组腺病毒感染后48h，以表达绿色荧光的细胞作为成功感染的细胞，SACC-83-WJ7＋WJ4组（沉默组）与SACC-83-HK组（空载体组）的感染率分别为98.7%和99.1%。 4Real-Time PCR检测XT-I和XT-II的沉默效果 感染后48h，SACC-83-WJ7＋WJ4组（沉默组）XT-I和XT-II的mRNA相对表达量明显低于SACC-83-HK组（空载体组）与SACC-83组（未感染组），SACC-83-HK组与SACC-83组之间无差别。SACC-83-WJ7＋WJ4组XT-I和XT-II的沉默率分别为97.3%和88.0%。 5XT-I和XT-II基因共沉默对SACC-83细胞PGs合成分泌的影响 感染后48h，SACC-83-WJ7＋WJ4组（沉默组）细胞培养液中PGs含量明显低于SACC-83-HK组（空载体组）和SACC-83组（未感染组），抑制率为68.61%，SACC-83-HK组PGs未受到抑制。 6裸鼠移植瘤最终体积 首次注射腺病毒后5周，，3组裸鼠移植瘤体积无统计学差异。 7流式细胞术测定裸鼠移植瘤细胞凋亡率 首次注射腺病毒后5周，3组裸鼠移植瘤细胞凋亡率无统计学差异。 8光学显微镜观察裸鼠移植瘤形态学变化 首次注射腺病毒后5周，光镜观察裸鼠移植瘤HE染色石蜡切片，3组间未见明显的形态学差异，均未观察到显著的细胞凋亡现象。 9透射电镜观察裸鼠移植瘤细胞凋亡现象 首次注射腺病毒后5周，SACC-83-WJ7＋WJ4组（沉默组）易见细胞凋亡现象，其余两组较少。 结论： 1重组腺病毒rAd5-shRNA-WJ7＋WJ4能够有效诱导SACC-83细胞XT-I和XT-II基因沉默，并阻抑PGs合成分泌。 2裸鼠SACC-83移植瘤内注射沉默XT-I和XT-II基因5周，沉默组出现细胞凋亡，但无统计学意义。
[Abstract]:Objective:
Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant salivary gland tumors. It is highly invasive, metastatic, prone to recurrence and poor prognosis. So far, there is no ideal treatment.
In SACC, neoplastic myoepithelial cells (NMCs) can secrete proteoglycans (PGs) to form extracellular matrix (ECM). In SACC, excessive ECM gradually accumulates to form cystic structure, thus showing the unique histological characteristics of SACC. Macromolecular microenvironment of SACC cells was studied. SACC cells survived in this microenvironment and progressed in a series of biological behaviors, such as proliferation, differentiation, invasion, metastasis and recurrence. Development process.
Human xylosyltransferase (XT) is the initial enzyme and rate-limiting enzyme in the biosynthesis of human PGs. Its activity truly reflects the biosynthesis rate of PGs. Human XT has two isoforms: XT-I and XT-II, which have almost the same catalytic activity, but different fineness in human body. The expression of cells was different.
RNA interference (RNAi) is currently the most effective gene silencing technology, which can specifically and efficiently block the expression of specific genes. The utilization rate of adenovirus vectors is high.
In this study, we intend to construct a plasmid vector that silences both XT-I and XT-II genes, i.e. two shRNA interference plasmids encoding recombinant adenovirus rAd5-shRNA-WJ7+WJ4, to interfere with the expression of XT-I and XT-II by infecting SACC-83 cells, to inhibit the synthesis of PGs, and to detect the silencing effect; to establish a nude mice transplanted tumor model of SACC-83. The aim of this study was to investigate the effects of XT-I and XT-II on PGs in SACC and the co-silencing of XT-I and XT-II on apoptosis of xenografts, and to provide more evidence for the biological treatment of salivary myoepithelial tumors.
Method:
1 construction of two recombinant plasmids targeting human XT-I gene and human XT-II gene respectively.
The shRNA-WJ4 targeting human XT-I gene and shRNA-WJ7 targeting human XT-II gene were designed. The shRNA-WJ4 and shRNA-WJ7 single-stranded target gene fragments were synthesized. The shRNA-WJ4 and shRNA-WJ7 fragments were linked to plasmids pGenesil-1.2 and pGenesil-1.1, amplified plasmids and identified by enzyme digestion. -shRNA-WJ4 and pGenesil-1.1-shRNA-WJ7.
2 construction of a co silencing plasmid targeting both human XT-I gene and human XT-II gene simultaneously.
Two or more recombinant plasmids were digested by double enzyme and the large and small fragments were recovered. The large fragment of WJ7 was linked to the small fragment of WJ4. The plasmids were amplified and identified by enzyme digestion. The shRNA-WJ7+WJ4 was sequenced to verify the co-silencing plasmid.
3 preparation of recombinant adenovirus plasmid
ShRNA-WJ7+WJ4 expression frame was transferred from plasmid pGenesil to pGSadeno adenovirus expression vector by LR homologous recombination in vitro. The plasmid was amplified and identified by enzyme digestion.
4 silence of recombinant adenovirus
The recombinant adenovirus rAd5-shRNA-WJ7+WJ4 was amplified and cultured in HEK293 cells.
5 construction of negative control recombinant adenovirus rAd5-shRNA-HK
6 recombinant adenovirus infected cells
SACC-83 cells were cultured and divided into three groups: SACC-83-WJ7+WJ4 group (silent group), SACC-83-HK group (empty carrier group), SACC-83 group (uninfected group). The optimal MOI value was 100, and the fluorescence intensity was the strongest after 48 hours of cell infection.
7 detect the expression of XT-I and XT-I mRNA in 3 groups after 48h infection.
Total RNA was extracted by TRIzol method, RNA purity, concentration, integrity, reverse transcription synthesis of the first strand of cDNA, real-time fluorescence quantitative PCR (Real-Time PCR) detection of three groups of cells XT-I and XT-I mRNA relative expression, calculate the silence rate.
8 detect the content of PGs in cell culture medium of 3 groups after 48h infection.
Standard absorbance standard curve was established by standard, PGs content was measured by biological staining, and inhibition rate was calculated.
Establishment and grouping of 9 nude mice xenograft models
Thirty-two four-week-old female BALB/c-nu nude mice, 14-15 g, were fed without specific pathogen-free (SPF) conditions, each of which was inoculated with SACC-83 cells 2.5 *106/250 Mu L on the left back. After 62 days, 24 BALB/c-nu nude mice with the longest diameter of more than 1 cm were randomly divided into three groups: SACC-83-WJ7+WJ4 group (silent group), SACC-83-HK group (empty group). Vector group, group SACC-83 (uninfected group), 8 in each group.
10 injection of recombinant adenovirus in nude mice
Sixty-two days after tumor cell inoculation, recombinant adenovirus was injected into the tumor for the first time. SACC-83-WJ7+WJ4 group (silent group) was injected with co-silent adenovirus rAd5-shRNA-WJ7+WJ4, SACC-83-HK group (empty vector group) was injected with negative control adenovirus rAd5-shRNA-HK, SACC-83 group (uninfected group) was injected with PBS, 4 *109 PFU/200 Mu once a week, five times.
11 specimen collection
Three groups of nude mice were sacrificed 5 weeks after the first injection of adenovirus. The transplanted tumors were separated and measured. The fresh tumors were immediately divided into 4 parts, 1 part was frozen at 80C, and 3 parts were used in the following experiments.
12 sample preparation and apoptosis detection by flow cytometry
Single cell suspension was prepared by 70% ethanol immobilization, splitting and mesh rubbing, and the apoptosis of the three groups was detected by PI staining and flow cytometry.
Preparation and observation of sample for 13HE dyeing
10% formalin fixed specimens, paraffin embedding, sections, HE staining, optical microscope to observe the morphological changes of the three groups of specimens.
14 preparation and observation of ultrathin section specimens of transmission electron microscope
Samples were cut to less than 1 mm 3,4% glutaraldehyde and fixed before 1% osmium acid. Epoxy resin was embedded. Ultra-thin sections were stained with uranium acetate and lead citrate for 30 minutes. Cell apoptosis was observed by transmission electron microscopy.
Result:
Sequence design of siRNA targeting XT-I, XT-II and negative control respectively
2 the results of enzyme digestion and agarose gel electrophoresis confirmed that the plasmid was correct.
3 the efficiency of recombinant adenovirus infecting SACC-83 cells.
At 48 hours after infection, the cells expressing green fluorescence were used as infected cells. The infection rates of SACC-83-WJ7+WJ4 group (silent group) and SACC-83-HK group (empty vector group) were 98.7% and 99.1% respectively.
Detection of XT-I and XT-II silencing effects by 4Real-Time PCR
At 48 hours after infection, the relative expression of XT-I and XT-II mRNA in SACC-83-WJ7+WJ4 group (silent group) was significantly lower than that in SACC-83-HK group (empty vector group) and SACC-83 group (uninfected group). There was no difference between SACC-83-HK group and SACC-83 group. The silencing rates of XT-I and XT-II in SACC-83-WJ7+WJ4 group were 97.3% and 88.0% respectively.
Effects of CO silencing of 5XT-I and XT-II gene on PGs synthesis and secretion in SACC-83 cells
At 48 hours after infection, PGs content in SACC-83-WJ7+WJ4 group (silent group) was significantly lower than that in SACC-83-HK group (empty carrier group) and SACC-83 group (non-infected group), the inhibition rate was 68.61%, and PGs in SACC-83-HK group was not inhibited.
6 final volume of transplanted tumor in nude mice
5 weeks after the first injection of adenovirus, there was no significant difference in the volume of transplanted tumor between the 3 groups.
7 the apoptosis rate of nude mice transplanted tumor cells was determined by flow cytometry.
5 weeks after the first injection of adenovirus, there was no significant difference in the apoptosis rate between the 3 groups.
8 the morphological changes of transplanted tumor in nude mice were observed by light microscope.
Five weeks after the first injection of adenovirus, the HE stained paraffin sections of xenografts in nude mice were observed under light microscope. There was no significant morphological difference among the three groups and no significant apoptosis was observed.
9 the apoptosis of transplanted tumor cells in nude mice was observed by transmission electron microscope.
Five weeks after the first injection of adenovirus, apoptosis was more common in SACC-83-WJ7+WJ4 group (silent group), but less in the other two groups.
Conclusion:
1 Recombinant adenovirus rAd5-shRNA-WJ7+WJ4 can effectively induce XT-I and XT-II gene silencing in SACC-83 cells and inhibit the synthesis and secretion of PGs.
XT-I and XT-II genes were silenced by injection into SACC-83 xenografts of nude mice for 5 weeks. Apoptosis was observed in the silent group, but there was no significant difference.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.87
本文编号：2182510
[Abstract]:Objective:
Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant salivary gland tumors. It is highly invasive, metastatic, prone to recurrence and poor prognosis. So far, there is no ideal treatment.
In SACC, neoplastic myoepithelial cells (NMCs) can secrete proteoglycans (PGs) to form extracellular matrix (ECM). In SACC, excessive ECM gradually accumulates to form cystic structure, thus showing the unique histological characteristics of SACC. Macromolecular microenvironment of SACC cells was studied. SACC cells survived in this microenvironment and progressed in a series of biological behaviors, such as proliferation, differentiation, invasion, metastasis and recurrence. Development process.
Human xylosyltransferase (XT) is the initial enzyme and rate-limiting enzyme in the biosynthesis of human PGs. Its activity truly reflects the biosynthesis rate of PGs. Human XT has two isoforms: XT-I and XT-II, which have almost the same catalytic activity, but different fineness in human body. The expression of cells was different.
RNA interference (RNAi) is currently the most effective gene silencing technology, which can specifically and efficiently block the expression of specific genes. The utilization rate of adenovirus vectors is high.
In this study, we intend to construct a plasmid vector that silences both XT-I and XT-II genes, i.e. two shRNA interference plasmids encoding recombinant adenovirus rAd5-shRNA-WJ7+WJ4, to interfere with the expression of XT-I and XT-II by infecting SACC-83 cells, to inhibit the synthesis of PGs, and to detect the silencing effect; to establish a nude mice transplanted tumor model of SACC-83. The aim of this study was to investigate the effects of XT-I and XT-II on PGs in SACC and the co-silencing of XT-I and XT-II on apoptosis of xenografts, and to provide more evidence for the biological treatment of salivary myoepithelial tumors.
Method:
1 construction of two recombinant plasmids targeting human XT-I gene and human XT-II gene respectively.
The shRNA-WJ4 targeting human XT-I gene and shRNA-WJ7 targeting human XT-II gene were designed. The shRNA-WJ4 and shRNA-WJ7 single-stranded target gene fragments were synthesized. The shRNA-WJ4 and shRNA-WJ7 fragments were linked to plasmids pGenesil-1.2 and pGenesil-1.1, amplified plasmids and identified by enzyme digestion. -shRNA-WJ4 and pGenesil-1.1-shRNA-WJ7.
2 construction of a co silencing plasmid targeting both human XT-I gene and human XT-II gene simultaneously.
Two or more recombinant plasmids were digested by double enzyme and the large and small fragments were recovered. The large fragment of WJ7 was linked to the small fragment of WJ4. The plasmids were amplified and identified by enzyme digestion. The shRNA-WJ7+WJ4 was sequenced to verify the co-silencing plasmid.
3 preparation of recombinant adenovirus plasmid
ShRNA-WJ7+WJ4 expression frame was transferred from plasmid pGenesil to pGSadeno adenovirus expression vector by LR homologous recombination in vitro. The plasmid was amplified and identified by enzyme digestion.
4 silence of recombinant adenovirus
The recombinant adenovirus rAd5-shRNA-WJ7+WJ4 was amplified and cultured in HEK293 cells.
5 construction of negative control recombinant adenovirus rAd5-shRNA-HK
6 recombinant adenovirus infected cells
SACC-83 cells were cultured and divided into three groups: SACC-83-WJ7+WJ4 group (silent group), SACC-83-HK group (empty carrier group), SACC-83 group (uninfected group). The optimal MOI value was 100, and the fluorescence intensity was the strongest after 48 hours of cell infection.
7 detect the expression of XT-I and XT-I mRNA in 3 groups after 48h infection.
Total RNA was extracted by TRIzol method, RNA purity, concentration, integrity, reverse transcription synthesis of the first strand of cDNA, real-time fluorescence quantitative PCR (Real-Time PCR) detection of three groups of cells XT-I and XT-I mRNA relative expression, calculate the silence rate.
8 detect the content of PGs in cell culture medium of 3 groups after 48h infection.
Standard absorbance standard curve was established by standard, PGs content was measured by biological staining, and inhibition rate was calculated.
Establishment and grouping of 9 nude mice xenograft models
Thirty-two four-week-old female BALB/c-nu nude mice, 14-15 g, were fed without specific pathogen-free (SPF) conditions, each of which was inoculated with SACC-83 cells 2.5 *106/250 Mu L on the left back. After 62 days, 24 BALB/c-nu nude mice with the longest diameter of more than 1 cm were randomly divided into three groups: SACC-83-WJ7+WJ4 group (silent group), SACC-83-HK group (empty group). Vector group, group SACC-83 (uninfected group), 8 in each group.
10 injection of recombinant adenovirus in nude mice
Sixty-two days after tumor cell inoculation, recombinant adenovirus was injected into the tumor for the first time. SACC-83-WJ7+WJ4 group (silent group) was injected with co-silent adenovirus rAd5-shRNA-WJ7+WJ4, SACC-83-HK group (empty vector group) was injected with negative control adenovirus rAd5-shRNA-HK, SACC-83 group (uninfected group) was injected with PBS, 4 *109 PFU/200 Mu once a week, five times.
11 specimen collection
Three groups of nude mice were sacrificed 5 weeks after the first injection of adenovirus. The transplanted tumors were separated and measured. The fresh tumors were immediately divided into 4 parts, 1 part was frozen at 80C, and 3 parts were used in the following experiments.
12 sample preparation and apoptosis detection by flow cytometry
Single cell suspension was prepared by 70% ethanol immobilization, splitting and mesh rubbing, and the apoptosis of the three groups was detected by PI staining and flow cytometry.
Preparation and observation of sample for 13HE dyeing
10% formalin fixed specimens, paraffin embedding, sections, HE staining, optical microscope to observe the morphological changes of the three groups of specimens.
14 preparation and observation of ultrathin section specimens of transmission electron microscope
Samples were cut to less than 1 mm 3,4% glutaraldehyde and fixed before 1% osmium acid. Epoxy resin was embedded. Ultra-thin sections were stained with uranium acetate and lead citrate for 30 minutes. Cell apoptosis was observed by transmission electron microscopy.
Result:
Sequence design of siRNA targeting XT-I, XT-II and negative control respectively
2 the results of enzyme digestion and agarose gel electrophoresis confirmed that the plasmid was correct.
3 the efficiency of recombinant adenovirus infecting SACC-83 cells.
At 48 hours after infection, the cells expressing green fluorescence were used as infected cells. The infection rates of SACC-83-WJ7+WJ4 group (silent group) and SACC-83-HK group (empty vector group) were 98.7% and 99.1% respectively.
Detection of XT-I and XT-II silencing effects by 4Real-Time PCR
At 48 hours after infection, the relative expression of XT-I and XT-II mRNA in SACC-83-WJ7+WJ4 group (silent group) was significantly lower than that in SACC-83-HK group (empty vector group) and SACC-83 group (uninfected group). There was no difference between SACC-83-HK group and SACC-83 group. The silencing rates of XT-I and XT-II in SACC-83-WJ7+WJ4 group were 97.3% and 88.0% respectively.
Effects of CO silencing of 5XT-I and XT-II gene on PGs synthesis and secretion in SACC-83 cells
At 48 hours after infection, PGs content in SACC-83-WJ7+WJ4 group (silent group) was significantly lower than that in SACC-83-HK group (empty carrier group) and SACC-83 group (non-infected group), the inhibition rate was 68.61%, and PGs in SACC-83-HK group was not inhibited.
6 final volume of transplanted tumor in nude mice
5 weeks after the first injection of adenovirus, there was no significant difference in the volume of transplanted tumor between the 3 groups.
7 the apoptosis rate of nude mice transplanted tumor cells was determined by flow cytometry.
5 weeks after the first injection of adenovirus, there was no significant difference in the apoptosis rate between the 3 groups.
8 the morphological changes of transplanted tumor in nude mice were observed by light microscope.
Five weeks after the first injection of adenovirus, the HE stained paraffin sections of xenografts in nude mice were observed under light microscope. There was no significant morphological difference among the three groups and no significant apoptosis was observed.
9 the apoptosis of transplanted tumor cells in nude mice was observed by transmission electron microscope.
Five weeks after the first injection of adenovirus, apoptosis was more common in SACC-83-WJ7+WJ4 group (silent group), but less in the other two groups.
Conclusion:
1 Recombinant adenovirus rAd5-shRNA-WJ7+WJ4 can effectively induce XT-I and XT-II gene silencing in SACC-83 cells and inhibit the synthesis and secretion of PGs.
XT-I and XT-II genes were silenced by injection into SACC-83 xenografts of nude mice for 5 weeks. Apoptosis was observed in the silent group, but there was no significant difference.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.87
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