【摘要】：目的: 唾液腺腺樣囊性癌（salivary adenoid cystic carcinoma, SACC）是最常見(jiàn)的唾液腺惡性腫瘤之一，侵襲性強(qiáng)，可轉(zhuǎn)移，易復(fù)發(fā)，預(yù)后不佳，迄今尚無(wú)較理想的治療措施。 SACC中的腫瘤性肌上皮細(xì)胞（neoplastic myoepithelial cells, NMCs）可以分泌蛋白多糖（proteoglycans, PGs），構(gòu)成腫瘤的細(xì)胞外基質(zhì)（extracellular matrix, ECM）。在SACC中，過(guò)多的ECM逐漸堆積，形成囊腔樣結(jié)構(gòu)，從而呈現(xiàn)出SACC特有的組織學(xué)特點(diǎn)。富含PGs的ECM，構(gòu)成了SACC細(xì)胞的大分子微環(huán)境（macromolecules microenvironment）。SACC細(xì)胞在這一微環(huán)境內(nèi)生存，繼而進(jìn)展，出現(xiàn)增殖、分化、侵襲、轉(zhuǎn)移、復(fù)發(fā)等一系列生物學(xué)行為。目前認(rèn)為，PGs是SACC的形態(tài)學(xué)表現(xiàn)和生物學(xué)行為必不可少的物質(zhì)基礎(chǔ)，參與腫瘤的發(fā)生和發(fā)展過(guò)程。 人木糖基轉(zhuǎn)移酶（xylosyltransferase, XT）是人體PGs生物合成的起始酶和限速酶，催化此合成過(guò)程的效率—限制階段（rate-limited step），它的活性真實(shí)反映PGs的生物合成率。人XT有兩個(gè)亞型（isoform）：XT-I和XT-II，催化作用幾乎相同，但是在人體不同細(xì)胞的表達(dá)有所差異。 RNA干擾（RNA interference, RNAi）是目前最有效的基因沉默（genesilencing）技術(shù)，可以特異、高效地阻斷特定基因的表達(dá)。腺病毒載體是將外源基因?qū)爰?xì)胞內(nèi)的常用媒介，同時(shí)表達(dá)一個(gè)以上目的基因的腺病毒載體，可同時(shí)發(fā)揮多個(gè)基因的功能，提高了腺病毒載體的利用率。 本研究擬構(gòu)建一個(gè)同時(shí)沉默XT-I和XT-II基因的質(zhì)粒載體，即編碼2條shRNA的干擾質(zhì)粒，腺病毒包裝，獲得重組腺病毒rAd5-shRNA-WJ7＋WJ4；通過(guò)感染SACC-83細(xì)胞，干擾XT-I和XT-II的表達(dá)，阻抑PGs合成，檢測(cè)其沉默效果；建立SACC-83的裸鼠移植瘤模型，瘤體內(nèi)注射重組腺病毒，觀察其對(duì)移植瘤凋亡的影響。旨在探討SACC中XT-I和XT-II對(duì)PGs的調(diào)控作用，以及XT-I和XT-II共沉默對(duì)細(xì)胞凋亡的影響，并為唾液腺肌上皮性腫瘤的生物治療提供更多的依據(jù)。 方法： 1構(gòu)建分別靶向抑制人XT-I基因和人XT-II基因的兩個(gè)重組質(zhì)粒 設(shè)計(jì)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的干擾位點(diǎn)和引物序列，合成shRNA-WJ4和shRNA-WJ7的單鏈目的基因片段，分別與質(zhì)粒pGenesil-1.2和pGenesil-1.1的線性化載體連接，擴(kuò)增質(zhì)粒，酶切鑒定，確定獲得重組質(zhì)粒pGenesil-1.2-shRNA-WJ4和pGenesil-1.1-shRNA-WJ7。 2構(gòu)建同時(shí)靶向抑制人XT-I基因和人XT-II基因的共沉默質(zhì)粒 雙酶切以上兩個(gè)重組質(zhì)粒，回收大小片段，WJ7大片段與WJ4小片段的連接，擴(kuò)增質(zhì)粒，酶切鑒定，測(cè)序驗(yàn)證共沉默質(zhì)粒shRNA-WJ7＋WJ4。 3重組腺病毒質(zhì)粒的制備 從質(zhì)粒pGenesil上通過(guò)LR體外同源重組將shRNA-WJ7＋WJ4表達(dá)框轉(zhuǎn)移至pGSadeno腺病毒表達(dá)載體上，擴(kuò)增質(zhì)粒，酶切鑒定。 4包裝共沉默重組腺病毒 制備線性化腺病毒DNA，連接重組腺病毒質(zhì)粒片段，轉(zhuǎn)染HEK293細(xì)胞，放大培養(yǎng)共沉默重組腺病毒rAd5-shRNA-WJ7＋WJ4。 5構(gòu)建陰性對(duì)照重組腺病毒rAd5-shRNA-HK 6重組腺病毒感染細(xì)胞 培養(yǎng)SACC-83細(xì)胞，分3組：SACC-83-WJ7＋WJ4組（沉默組）、SACC-83-HK組（空載體組）、SACC-83組（未感染組），確定最佳MOI值為100，細(xì)胞感染48h的熒光最強(qiáng)，計(jì)算感染率。 7檢測(cè)感染48h后3組細(xì)胞XT-I和XT-I mRNA的表達(dá) TRIzol法提取細(xì)胞總RNA，測(cè)定RNA純度、濃度、完整性，逆轉(zhuǎn)錄合成cDNA第一鏈，實(shí)時(shí)熒光定量PCR（Real-Time PCR）檢測(cè)3組細(xì)胞XT-I和XT-I mRNA的相對(duì)表達(dá)量，計(jì)算沉默率。 8檢測(cè)感染48h后3組細(xì)胞培養(yǎng)液中PGs的含量 用標(biāo)準(zhǔn)品建立濃度-吸光度標(biāo)準(zhǔn)曲線，生物染色法測(cè)定PGs含量，計(jì)算抑制率。 9裸鼠移植瘤模型的建立及分組 4周齡雌性BALB/c-nu裸小鼠32只，14～15g，無(wú)特定病原體（specificpathogen-free, SPF）條件飼養(yǎng)，每只于左側(cè)背部接種SACC-83細(xì)胞2.5×106個(gè)／250μl；62d后，選取瘤體最長(zhǎng)徑在1cm以上的24只，隨機(jī)數(shù)字表法分為3組：SACC-83-WJ7＋WJ4組（沉默組）、SACC-83-HK組（空載體組）、SACC-83組（未感染組），每組8只。 10重組腺病毒裸鼠移植瘤內(nèi)注射 接種腫瘤細(xì)胞后62d，首次瘤內(nèi)注射重組腺病毒，SACC-83-WJ7＋WJ4組（沉默組）注射共沉默腺病毒rAd5-shRNA-WJ7＋WJ4，SACC-83-HK組（空載體組）注射陰性對(duì)照腺病毒rAd5-shRNA-HK，SACC-83組（未感染組）注射PBS，每只4×109PFU／200μl，每周注射一次，共5次。 11標(biāo)本采集 3組裸鼠首次注射腺病毒5周后斷頸處死，分離移植瘤，測(cè)量體積，新鮮瘤組織立即分切為4份，1份80C凍存，3份用于以下實(shí)驗(yàn)。 12流式細(xì)胞術(shù)樣品制備和凋亡檢測(cè) 70%乙醇固定標(biāo)本，剪碎法＋網(wǎng)搓法制備單細(xì)胞懸液，PI染色，流式細(xì)胞術(shù)檢測(cè)3組樣品的細(xì)胞凋亡情況。 13HE染色樣品制備和觀察 10%福爾馬林固定標(biāo)本、石蠟包埋、切片、HE染色、光學(xué)顯微鏡觀察3組標(biāo)本的形態(tài)學(xué)變化。 14透射電鏡超薄切片樣品制備和觀察 標(biāo)本分切至小于1mm3，4%戊二醛前固定、1%鋨酸后固定、環(huán)氧樹脂包埋、超薄切片、醋酸鈾和檸檬酸鉛雙重染色30min、透射電鏡觀察細(xì)胞凋亡情況。 結(jié)果： 1分別靶向XT-I、XT-II和陰性對(duì)照的siRNA序列設(shè)計(jì)結(jié)果shRNA-WJ4CAGGCAGCCCATCAAACCTshRNA-WJ7CGTCTCCTCAAGGCCGTTTATshRNA-HK GACTTCATAAGGCGCATGC 2酶切和瓊脂糖凝膠電泳鑒定結(jié)果、測(cè)序驗(yàn)證結(jié)果均證明質(zhì)粒正確。 3重組腺病毒感染SACC-83細(xì)胞的效率 重組腺病毒感染后48h，以表達(dá)綠色熒光的細(xì)胞作為成功感染的細(xì)胞，SACC-83-WJ7＋WJ4組（沉默組）與SACC-83-HK組（空載體組）的感染率分別為98.7%和99.1%。 4Real-Time PCR檢測(cè)XT-I和XT-II的沉默效果 感染后48h，SACC-83-WJ7＋WJ4組（沉默組）XT-I和XT-II的mRNA相對(duì)表達(dá)量明顯低于SACC-83-HK組（空載體組）與SACC-83組（未感染組），SACC-83-HK組與SACC-83組之間無(wú)差別。SACC-83-WJ7＋WJ4組XT-I和XT-II的沉默率分別為97.3%和88.0%。 5XT-I和XT-II基因共沉默對(duì)SACC-83細(xì)胞PGs合成分泌的影響 感染后48h，SACC-83-WJ7＋WJ4組（沉默組）細(xì)胞培養(yǎng)液中PGs含量明顯低于SACC-83-HK組（空載體組）和SACC-83組（未感染組），抑制率為68.61%，SACC-83-HK組PGs未受到抑制。 6裸鼠移植瘤最終體積 首次注射腺病毒后5周，，3組裸鼠移植瘤體積無(wú)統(tǒng)計(jì)學(xué)差異。 7流式細(xì)胞術(shù)測(cè)定裸鼠移植瘤細(xì)胞凋亡率 首次注射腺病毒后5周，3組裸鼠移植瘤細(xì)胞凋亡率無(wú)統(tǒng)計(jì)學(xué)差異。 8光學(xué)顯微鏡觀察裸鼠移植瘤形態(tài)學(xué)變化 首次注射腺病毒后5周，光鏡觀察裸鼠移植瘤HE染色石蠟切片，3組間未見(jiàn)明顯的形態(tài)學(xué)差異，均未觀察到顯著的細(xì)胞凋亡現(xiàn)象。 9透射電鏡觀察裸鼠移植瘤細(xì)胞凋亡現(xiàn)象 首次注射腺病毒后5周，SACC-83-WJ7＋WJ4組（沉默組）易見(jiàn)細(xì)胞凋亡現(xiàn)象，其余兩組較少。 結(jié)論： 1重組腺病毒rAd5-shRNA-WJ7＋WJ4能夠有效誘導(dǎo)SACC-83細(xì)胞XT-I和XT-II基因沉默，并阻抑PGs合成分泌。 2裸鼠SACC-83移植瘤內(nèi)注射沉默XT-I和XT-II基因5周，沉默組出現(xiàn)細(xì)胞凋亡，但無(wú)統(tǒng)計(jì)學(xué)意義。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.87

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