步(bu)(bu)(bu)(bu)驟i.將(jiang)步(bu)(bu)(bu)(bu)驟h的吸附柱放入收集管，12000rpm離心(xin)(xin)2min。步(bu)(bu)(bu)(bu)驟j.吸附柱放到一(yi)個新的，在吸附柱膜**加(jia)(jia)(jia)入50～200μl滅菌水(shui)或者洗(xi)脫液(ye)，停留5min。(將(jiang)無菌水(shui)或者洗(xi)脫液(ye)加(jia)(jia)(jia)熱到65℃能夠增(zeng)(zeng)加(jia)(jia)(jia)洗(xi)脫的得(de)率)。步(bu)(bu)(bu)(bu)驟i.基(ji)因組(zu)(zu)dna定量，洗(xi)脫的基(ji)因組(zu)(zu)dna可(ke)以通過(guo)電(dian)泳鑒(jian)定。方法(fa)2：一(yi)種低(di)成本基(ji)因組(zu)(zu)dna的粗提方法(fa)：步(bu)(bu)(bu)(bu)驟a.每(mei)只小鼠剪下一(yi)段(duan)尾巴(ba)(2-5mm)，放入離心(xin)(xin)管中，加(jia)(jia)(jia)入100μl尾部(bu)消(xiao)(xiao)化緩(huan)沖液(ye)，注意(yi)不要剪尾太長；步(bu)(bu)(bu)(bu)驟b.將(jiang)離心(xin)(xin)管及(ji)其內容物(wu)(wu)于56℃孵(fu)育(yu)過(guo)夜；步(bu)(bu)(bu)(bu)驟c.過(guo)夜后將(jiang)離心(xin)(xin)管于98℃孵(fu)育(yu)13min，使(shi)(shi)蛋白酶k失(shi)活；步(bu)(bu)(bu)(bu)驟d.在微(wei)型離心(xin)(xin)機以**大轉(zhuan)速(su)旋轉(zhuan)15min，上(shang)清直接用(yong)于pcr體(ti)(ti)系(每(mei)50μlpcr體(ti)(ti)系加(jia)(jia)(jia)入上(shang)清2μl)。尾消(xiao)(xiao)化緩(huan)沖液(ye)成分：50mmkcl10mmtris-hcl()％tritonx-100(b)長鏈pcr反(fan)應(ying)：長鏈引物(wu)(wu)：pcrprimers1(℃):擴增(zeng)(zeng)片段(duan)：5’armforwardprimer(f1):5’-tacgccacagggagtccaagaatg-3’5’kireverseprimer(r1):5’-gatggggagagtgaagcagaacg-3’pcrprimers2(℃):擴增(zeng)(zeng)片段(duan)：3’kiforwardprimer(f2):5’-ctgctgtccattccttattccatag-3’3’armreverseprimer(r2):5’-ctggaaatcaggctgcaaatctc-3’pirb基(ji)因敲除型有(you)上(shang)述兩條擴增(zeng)(zeng)片段(duan)，野生(sheng)型等位基(ji)因沒有(you)擴增(zeng)(zeng)產物(wu)(wu)。人(ren)類疾(ji)病的動物(wu)(wu)模型是指各(ge)種醫(yi)學科學研究中建立(li)的具有(you)人(ren)類疾(ji)病模擬表現的動物(wu)(wu)。徐州如何使(shi)(shi)用(yong)疾(ji)病動物(wu)(wu)模型建模

1、動物(wu)(wu)模(mo)(mo)型(xing)(xing)名稱(cheng)：堿燒傷(shang)致角膜(mo)損傷(shang)大(da)鼠(shu)(shu)模(mo)(mo)型(xing)(xing)2、實驗動物(wu)(wu)種屬：SD大(da)鼠(shu)(shu)3、實驗動物(wu)(wu)性別：雌(ci)雄不限4、實驗動物(wu)(wu)年(nian)齡：成(cheng)年(nian)5、實驗動物(wu)(wu)體重：160-200g6、實驗動物(wu)(wu)環(huan)境：SPF級，1、實驗方法：氫(qing)氧化(hua)鈉致大(da)鼠(shu)(shu)角膜(mo)化(hua)學性損傷(shang)。大(da)鼠(shu)(shu)用戊(wu)巴比妥鈉腹腔注射麻(ma)醉，用干(gan)(gan)棉棒拭去(qu)結(jie)膜(mo)囊多余(yu)液體，將統一規格直徑3mm的圓形濾紙(zhi)(zhi)片浸(jin)入1mol/LNaOH溶液中20s，飽和后(hou)(hou)取(qu)出置于干(gan)(gan)燥濾紙(zhi)(zhi)上1秒以(yi)吸去(qu)過多的堿液，將濾紙(zhi)(zhi)片貼敷于大(da)鼠(shu)(shu)右眼角膜(mo)**90s后(hou)(hou)移(yi)去(qu)，立(li)即(ji)用滅菌水沖洗(xi)結(jie)膜(mo)囊及角膜(mo)2min。2、檢測標準(zhun)：肉(rou)眼或裂隙燈下觀察到(dao)角膜(mo)有損傷(shang)。提供(gong)疾病(bing)動物(wu)(wu)模(mo)(mo)型(xing)(xing)建模(mo)(mo)公司通(tong)過小鼠(shu)(shu)疾病(bing)模(mo)(mo)型(xing)(xing)的構建有助于我(wo)們深入揭(jie)示潛在致病(bing)基(ji)因作用機制。

本能(neng)發(fa)明(ming)(ming)(ming)的(de)(de)(de)另一(yi)目的(de)(de)(de)是公開了如上(shang)所述的(de)(de)(de)方(fang)(fang)(fang)法(fa)(fa)制(zhi)備(bei)的(de)(de)(de)鼠慢性背根神(shen)經(jing)壓(ya)(ya)迫(po)(po)(po)(po)模(mo)(mo)型(xing)的(de)(de)(de)用(yong)(yong)(yong)途(tu)是將該模(mo)(mo)型(xing)用(yong)(yong)(yong)于(yu)(yu)腰(yao)椎(zhui)間(jian)盤(pan)(pan)突(tu)(tu)出(chu)(chu)方(fang)(fang)(fang)法(fa)(fa)研究(jiu)(jiu)(jiu)和(he)/或(huo)影(ying)像學(xue)檢(jian)測研究(jiu)(jiu)(jiu)。進(jin)(jin)一(yi)步(bu)地，該模(mo)(mo)型(xing)用(yong)(yong)(yong)于(yu)(yu)與磁(ci)(ci)性相關的(de)(de)(de)和(he)/或(huo)檢(jian)測方(fang)(fang)(fang)面的(de)(de)(de)研究(jiu)(jiu)(jiu)，比如經(jing)顱磁(ci)(ci)刺(ci)激和(he)核磁(ci)(ci)共(gong)(gong)振。本發(fa)明(ming)(ming)(ming)利(li)用(yong)(yong)(yong)壓(ya)(ya)迫(po)(po)(po)(po)元件(jian)插入腰(yao)椎(zhui)椎(zhui)間(jian)孔，模(mo)(mo)擬腰(yao)椎(zhui)間(jian)盤(pan)(pan)突(tu)(tu)出(chu)(chu)后突(tu)(tu)出(chu)(chu)物(wu)(wu)對(dui)(dui)神(shen)經(jing)根的(de)(de)(de)持續慢性的(de)(de)(de)機械(xie)壓(ya)(ya)迫(po)(po)(po)(po)癥(zheng)狀(zhuang)(zhuang)，有益效果有：1.應用(yong)(yong)(yong)機械(xie)壓(ya)(ya)迫(po)(po)(po)(po)神(shen)經(jing)根模(mo)(mo)擬腰(yao)椎(zhui)間(jian)盤(pan)(pan)突(tu)(tu)出(chu)(chu)病理，癥(zheng)狀(zhuang)(zhuang)的(de)(de)(de)病因學(xue)與臨(lin)床(chuang)情況接近；2.相關的(de)(de)(de)疼(teng)痛行(xing)為(wei)類似于(yu)(yu)特定的(de)(de)(de)慢性疼(teng)痛癥(zheng)狀(zhuang)(zhuang)，且(qie)易于(yu)(yu)客觀測量；3.制(zhi)備(bei)方(fang)(fang)(fang)法(fa)(fa)簡單(dan)，對(dui)(dui)動(dong)物(wu)(wu)損(sun)傷小，穩定性好，成(cheng)功(gong)率高；4.造模(mo)(mo)關鍵材(cai)料(liao)壓(ya)(ya)迫(po)(po)(po)(po)元件(jian)不受磁(ci)(ci)場(chang)(chang)干擾，無在(zai)磁(ci)(ci)場(chang)(chang)作用(yong)(yong)(yong)下移位風險，有利(li)于(yu)(yu)后續對(dui)(dui)動(dong)物(wu)(wu)進(jin)(jin)行(xing)磁(ci)(ci)療，經(jing)顱磁(ci)(ci)刺(ci)激等(deng)(deng)及核磁(ci)(ci)共(gong)(gong)振等(deng)(deng)檢(jian)查；5.造模(mo)(mo)關鍵材(cai)料(liao)壓(ya)(ya)迫(po)(po)(po)(po)元件(jian)不影(ying)響(xiang)磁(ci)(ci)場(chang)(chang)，不會對(dui)(dui)儀器和(he)核磁(ci)(ci)共(gong)(gong)振等(deng)(deng)儀器產生不良影(ying)響(xiang)；6.此方(fang)(fang)(fang)法(fa)(fa)獲得的(de)(de)(de)模(mo)(mo)型(xing)由于(yu)(yu)采(cai)用(yong)(yong)(yong)了h62黃銅或(huo)聚(ju)乳酸材(cai)料(liao)制(zhi)備(bei)的(de)(de)(de)壓(ya)(ya)迫(po)(po)(po)(po)元件(jian)，使得研究(jiu)(jiu)(jiu)方(fang)(fang)(fang)法(fa)(fa)與檢(jian)測方(fang)(fang)(fang)法(fa)(fa)不受磁(ci)(ci)場(chang)(chang)作用(yong)(yong)(yong)限制(zhi)，豐富了腰(yao)椎(zhui)間(jian)盤(pan)(pan)突(tu)(tu)出(chu)(chu)臨(lin)床(chuang)前研究(jiu)(jiu)(jiu)的(de)(de)(de)方(fang)(fang)(fang)法(fa)(fa)及影(ying)像學(xue)檢(jian)測，為(wei)臨(lin)床(chuang)研究(jiu)(jiu)(jiu)提供理論依據。以下將結合附(fu)(fu)圖對(dui)(dui)本發(fa)明(ming)(ming)(ming)作進(jin)(jin)一(yi)步(bu)說明(ming)(ming)(ming)，以充分說明(ming)(ming)(ming)本發(fa)明(ming)(ming)(ming)的(de)(de)(de)目的(de)(de)(de)、技術(shu)特征和(he)技術(shu)效果。附(fu)(fu)圖說明(ming)(ming)(ming)圖1是壓(ya)(ya)迫(po)(po)(po)(po)元件(jian)插入椎(zhui)間(jian)孔的(de)(de)(de)結構示意圖。

1、動(dong)(dong)物(wu)(wu)模(mo)型名(ming)稱：皮(pi)(pi)(pi)膚(fu)淺(qian)(qian)II°及深II°燙(tang)(tang)傷(shang)(shang)大鼠(shu)(shu)(shu)模(mo)型2、實(shi)(shi)驗(yan)(yan)動(dong)(dong)物(wu)(wu)種屬：SD大鼠(shu)(shu)(shu)3、實(shi)(shi)驗(yan)(yan)動(dong)(dong)物(wu)(wu)性(xing)別：雌雄不限4、實(shi)(shi)驗(yan)(yan)動(dong)(dong)物(wu)(wu)年齡：成年5、實(shi)(shi)驗(yan)(yan)動(dong)(dong)物(wu)(wu)體重：160-200g6、實(shi)(shi)驗(yan)(yan)動(dong)(dong)物(wu)(wu)環境：SPF級1、實(shi)(shi)驗(yan)(yan)方法：熱力致皮(pi)(pi)(pi)膚(fu)損傷(shang)(shang)法。麻(ma)醉大鼠(shu)(shu)(shu)，根據體重腹部備皮(pi)(pi)(pi)，然(ran)后固定于實(shi)(shi)驗(yan)(yan)臺上。將大鼠(shu)(shu)(shu)移近加熱純(chun)凈(jing)水(shui)的燒杯，將紗布條浸入熱水(shui)中，待水(shui)溫恒(heng)定于99℃時，取出(chu)紗布，立(li)即(ji)平鋪于待燙(tang)(tang)部位，于紗布接(jie)觸大鼠(shu)(shu)(shu)皮(pi)(pi)(pi)膚(fu)后開始(shi)計時。致傷(shang)(shang)3s為淺(qian)(qian)II°燙(tang)(tang)傷(shang)(shang)，致傷(shang)(shang)10s為深II°燙(tang)(tang)傷(shang)(shang)。2、檢(jian)測標準：a.皮(pi)(pi)(pi)膚(fu)大體觀察：致傷(shang)(shang)3s大鼠(shu)(shu)(shu)局部皮(pi)(pi)(pi)膚(fu)發(fa)紅、輕(qing)微(wei)水(shui)腫。愈(yu)合后毛(mao)發(fa)生長良(liang)好(hao)，有少量紅褐**素沉著，皮(pi)(pi)(pi)膚(fu)輕(qing)微(wei)攣縮，表皮(pi)(pi)(pi)光滑；致傷(shang)(shang)10s大鼠(shu)(shu)(shu)局部皮(pi)(pi)(pi)膚(fu)發(fa)白、水(shui)腫。愈(yu)合后毛(mao)發(fa)生長良(liang)好(hao)，皮(pi)(pi)(pi)膚(fu)瘢痕形成，表面粗糙不平。構(gou)建人源移植瘤模(mo)型需要(yao)合適(shi)的免疫缺陷(xian)小鼠(shu)(shu)(shu)想知道如何選(xuan)擇？

因(yin)此，應(ying)用(yong)動(dong)物(wu)模型，除了能克(ke)(ke)服在(zai)人(ren)(ren)(ren)類(lei)(lei)研究(jiu)(jiu)中經常會(hui)遇到的(de)(de)(de)理論和(he)社會(hui)限制外，還容(rong)許采(cai)用(yong)某(mou)些不能應(ying)用(yong)于人(ren)(ren)(ren)類(lei)(lei)的(de)(de)(de)方(fang)法學途徑，甚至為了研究(jiu)(jiu)需要(yao)可(ke)以(yi)損傷動(dong)物(wu)組織、或處死(si)動(dong)物(wu)。（二）臨床(chuang)上(shang)平時(shi)不易見(jian)到的(de)(de)(de)疾病(bing)(bing)(bing)(bing)可(ke)用(yong)動(dong)物(wu)隨時(shi)復(fu)制出(chu)來臨床(chuang)上(shang)平時(shi)很(hen)難(nan)收集到放射(she)病(bing)(bing)(bing)(bing)、毒氣中毒、烈性(xing)傳(chuan)染病(bing)(bing)(bing)(bing)等病(bing)(bing)(bing)(bing)人(ren)(ren)(ren)，而(er)(er)實(shi)驗室(shi)可(ke)以(yi)根據(ju)研究(jiu)(jiu)目(mu)的(de)(de)(de)要(yao)求隨時(shi)采(cai)用(yong)實(shi)驗性(xing)誘發(fa)的(de)(de)(de)方(fang)法在(zai)動(dong)物(wu)身上(shang)復(fu)制出(chu)來。（三）可(ke)以(yi)克(ke)(ke)服人(ren)(ren)(ren)類(lei)(lei)某(mou)些疾病(bing)(bing)(bing)(bing)潛(qian)伏(fu)期長，病(bing)(bing)(bing)(bing)程(cheng)長和(he)發(fa)病(bing)(bing)(bing)(bing)率(lv)低(di)的(de)(de)(de)缺點一般遺傳(chuan)性(xing)、免疫(yi)性(xing)、代謝(xie)性(xing)和(he)內分泌等疾病(bing)(bing)(bing)(bing)在(zai)臨床(chuang)上(shang)發(fa)病(bing)(bing)(bing)(bing)率(lv)很(hen)低(di)，例如急性(xing)白(bai)血病(bing)(bing)(bing)(bing)的(de)(de)(de)發(fa)病(bing)(bing)(bing)(bing)率(lv)較降，研究(jiu)(jiu)人(ren)(ren)(ren)員可(ke)以(yi)有意識(shi)地(di)提高其(qi)在(zai)動(dong)物(wu)種(zhong)群的(de)(de)(de)中發(fa)生頻率(lv)，從而(er)(er)推進(jin)研究(jiu)(jiu)。同(tong)樣的(de)(de)(de)途徑已(yi)成功地(di)應(ying)用(yong)于其(qi)他(ta)疾病(bing)(bing)(bing)(bing)的(de)(de)(de)研究(jiu)(jiu)，如血友病(bing)(bing)(bing)(bing)、周期性(xing)中性(xing)白(bai)細胞減少癥和(he)自(zi)身免疫(yi)介導(dao)性(xing)疾病(bing)(bing)(bing)(bing)等。動(dong)物(wu)疾病(bing)(bing)(bing)(bing)模型廣泛(fan)應(ying)用(yong)于疾病(bing)(bing)(bing)(bing)機制研究(jiu)(jiu)。徐州正(zheng)規疾病(bing)(bing)(bing)(bing)動(dong)物(wu)模型建模

動(dong)物(wu)疾(ji)病模(mo)型的另(ling)一個富有(you)成效的用(yong)途。徐(xu)州如何(he)使用(yong)疾(ji)病動(dong)物(wu)模(mo)型建模(mo)

轉(zhuan)(zhuan)(zhuan)基(ji)(ji)(ji)因(yin)(yin)小(xiao)(xiao)(xiao)鼠(shu)對(dui)黑(hei)(hei)色(se)(se)素瘤(liu)(liu)(liu)發生的(de)(de)(de)(de)分子(zi)途(tu)徑(jing)的(de)(de)(de)(de)理解有(you)重(zhong)(zhong)要意(yi)義。此外，轉(zhuan)(zhuan)(zhuan)基(ji)(ji)(ji)因(yin)(yin)小(xiao)(xiao)(xiao)鼠(shu)是(shi)(shi)(shi)可(ke)靠(kao)且可(ke)重(zhong)(zhong)現的(de)(de)(de)(de)模(mo)型(xing)(xing)，用(yong)來評估受損基(ji)(ji)(ji)因(yin)(yin)對(dui)黑(hei)(hei)色(se)(se)素瘤(liu)(liu)(liu)生物(wu)(wu)學的(de)(de)(de)(de)影響。應用(yong)：基(ji)(ji)(ji)因(yin)(yin)工程小(xiao)(xiao)(xiao)鼠(shu)模(mo)型(xing)(xing)可(ke)用(yong)于(yu)研究(jiu)(jiu)特定基(ji)(ji)(ji)因(yin)(yin)組(zu)改變在黑(hei)(hei)色(se)(se)素瘤(liu)(liu)(liu)的(de)(de)(de)(de)發生和(he)發展的(de)(de)(de)(de)重(zhong)(zhong)要性及藥物(wu)(wu)的(de)(de)(de)(de)靶向。1Lum－/－基(ji)(ji)(ji)因(yin)(yin)敲除(chu)小(xiao)(xiao)(xiao)鼠(shu)Lumican是(shi)(shi)(shi)一種富(fu)含亮(liang)氨酸(suan)的(de)(de)(de)(de)小(xiao)(xiao)(xiao)蛋白(bai)聚糖(tang)(smallleucine-richproteoglycan，SLRP)，為(wei)膠原原纖維形成(cheng)(cheng)的(de)(de)(de)(de)關鍵調(diao)節劑。黑(hei)(hei)色(se)(se)素瘤(liu)(liu)(liu)中Lumican表達降低與(yu)浸(jin)潤(run)相(xiang)關。方法：有(you)研究(jiu)(jiu)者(zhe)利用(yong)BamHI建立的(de)(de)(de)(de)克隆(long)衍生物(wu)(wu)導入胚胎干細(xi)(xi)胞，建立Lum－/－轉(zhuan)(zhuan)(zhuan)基(ji)(ji)(ji)因(yin)(yin)小(xiao)(xiao)(xiao)鼠(shu)。該模(mo)型(xing)(xing)可(ke)提供與(yu)發生和(he)轉(zhuan)(zhuan)(zhuan)移相(xiang)關機制及可(ke)見(jian)變的(de)(de)(de)(de)進展，以及確定負責的(de)(de)(de)(de)黑(hei)(hei)色(se)(se)素瘤(liu)(liu)(liu)進展的(de)(de)(de)(de)基(ji)(ji)(ji)因(yin)(yin)。2Tyr::N-RasQ61K轉(zhuan)(zhuan)(zhuan)基(ji)(ji)(ji)因(yin)(yin)小(xiao)(xiao)(xiao)鼠(shu)方法：有(you)研究(jiu)(jiu)者(zhe)使(shi)(shi)(shi)用(yong)突變的(de)(de)(de)(de)人(ren)N-ＲasQ61K和(he)SV40剪(jian)接(jie)以及聚腺苷酸(suan)化序(xu)列(lie)生成(cheng)(cheng)Tyr::N-ＲasQ61K構建體，并(bing)注射(she)到卵母細(xi)(xi)胞中，建立Tyr::N-ＲasQ61K轉(zhuan)(zhuan)(zhuan)基(ji)(ji)(ji)因(yin)(yin)小(xiao)(xiao)(xiao)鼠(shu)。3BrafV600E轉(zhuan)(zhuan)(zhuan)基(ji)(ji)(ji)因(yin)(yin)小(xiao)(xiao)(xiao)鼠(shu)模(mo)型(xing)(xing)有(you)研究(jiu)(jiu)者(zhe)使(shi)(shi)(shi)用(yong)LoxP-stop-LoxP(LSL)/Cre重(zhong)(zhong)組(zu)酶技術從內(nei)源性Braf基(ji)(ji)(ji)因(yin)(yin)中誘(you)導BrafV600E表達，建立BrafV600E轉(zhuan)(zhuan)(zhuan)基(ji)(ji)(ji)因(yin)(yin)黑(hei)(hei)色(se)(se)素瘤(liu)(liu)(liu)小(xiao)(xiao)(xiao)鼠(shu)模(mo)型(xing)(xing)。總結(jie)目前已有(you)三種不同的(de)(de)(de)(de)黑(hei)(hei)色(se)(se)素瘤(liu)(liu)(liu)小(xiao)(xiao)(xiao)鼠(shu)模(mo)型(xing)(xing)，但由于(yu)實驗(yan)動物(wu)(wu)與(yu)人(ren)類基(ji)(ji)(ji)因(yin)(yin)組(zu)、基(ji)(ji)(ji)因(yin)(yin)調(diao)控、細(xi)(xi)胞類型(xing)(xing)、組(zu)成(cheng)(cheng)等方面是(shi)(shi)(shi)有(you)一定差別。徐州如何使(shi)(shi)(shi)用(yong)疾病動物(wu)(wu)模(mo)型(xing)(xing)建模(mo)

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