步(bu)骤(zhou)i.将(jiang)步(bu)骤(zhou)h的(de)(de)吸(xi)附(fu)(fu)(fu)柱放入(ru)(ru)(ru)(ru)收集管，12000rpm离心(xin)2min。步(bu)骤(zhou)j.吸(xi)附(fu)(fu)(fu)柱放到(dao)(dao)一(yi)(yi)个新的(de)(de)，在(zai)吸(xi)附(fu)(fu)(fu)柱膜**加(jia)入(ru)(ru)(ru)(ru)50～200μl灭菌水(shui)或(huo)者洗(xi)脱(tuo)液，停留(liu)5min。(将(jiang)无(wu)菌水(shui)或(huo)者洗(xi)脱(tuo)液加(jia)热(re)到(dao)(dao)65℃能够增(zeng)(zeng)加(jia)洗(xi)脱(tuo)的(de)(de)得(de)率(lv))。步(bu)骤(zhou)i.基(ji)因(yin)组(zu)dna定(ding)量，洗(xi)脱(tuo)的(de)(de)基(ji)因(yin)组(zu)dna可以通过电泳鉴(jian)定(ding)。方法2：一(yi)(yi)种低成本基(ji)因(yin)组(zu)dna的(de)(de)粗提方法：步(bu)骤(zhou)a.每只小(xiao)鼠剪下一(yi)(yi)段(duan)尾(wei)巴(2-5mm)，放入(ru)(ru)(ru)(ru)离心(xin)管中(zhong)，加(jia)入(ru)(ru)(ru)(ru)100μl尾(wei)部消化缓冲(chong)液，注意(yi)不要剪尾(wei)太(tai)长(zhang)；步(bu)骤(zhou)b.将(jiang)离心(xin)管及其内容物于(yu)56℃孵育过夜；步(bu)骤(zhou)c.过夜后将(jiang)离心(xin)管于(yu)98℃孵育13min，使蛋白酶k失活；步(bu)骤(zhou)d.在(zai)微型(xing)(xing)(xing)离心(xin)机以**大转(zhuan)速旋转(zhuan)15min，上清直接用于(yu)pcr体系(xi)(每50μlpcr体系(xi)加(jia)入(ru)(ru)(ru)(ru)上清2μl)。尾(wei)消化缓冲(chong)液成分(fen)：50mmkcl10mmtris-hcl()％tritonx-100(b)长(zhang)链pcr反应(ying)：长(zhang)链引物：pcrprimers1(℃):扩增(zeng)(zeng)片(pian)段(duan)：5’armforwardprimer(f1):5’-tacgccacagggagtccaagaatg-3’5’kireverseprimer(r1):5’-gatggggagagtgaagcagaacg-3’pcrprimers2(℃):扩增(zeng)(zeng)片(pian)段(duan)：3’kiforwardprimer(f2):5’-ctgctgtccattccttattccatag-3’3’armreverseprimer(r2):5’-ctggaaatcaggctgcaaatctc-3’pirb基(ji)因(yin)敲除型(xing)(xing)(xing)有(you)上述两(liang)条(tiao)扩增(zeng)(zeng)片(pian)段(duan)，野生型(xing)(xing)(xing)等位(wei)基(ji)因(yin)没有(you)扩增(zeng)(zeng)产物。人类(lei)(lei)疾病(bing)的(de)(de)动物模型(xing)(xing)(xing)是指各种医学科学研究中(zhong)建立(li)的(de)(de)具有(you)人类(lei)(lei)疾病(bing)模拟表现的(de)(de)动物。徐州如何使用疾病(bing)动物模型(xing)(xing)(xing)建模

1、动(dong)(dong)物(wu)(wu)模(mo)型(xing)(xing)名称：碱烧(shao)伤致(zhi)角(jiao)膜(mo)损(sun)伤大鼠(shu)模(mo)型(xing)(xing)2、实(shi)验(yan)(yan)动(dong)(dong)物(wu)(wu)种属：SD大鼠(shu)3、实(shi)验(yan)(yan)动(dong)(dong)物(wu)(wu)性(xing)别：雌雄不限(xian)4、实(shi)验(yan)(yan)动(dong)(dong)物(wu)(wu)年龄：成年5、实(shi)验(yan)(yan)动(dong)(dong)物(wu)(wu)体重(zhong)：160-200g6、实(shi)验(yan)(yan)动(dong)(dong)物(wu)(wu)环境：SPF级，1、实(shi)验(yan)(yan)方法：氢氧(yang)化钠致(zhi)大鼠(shu)角(jiao)膜(mo)化学性(xing)损(sun)伤。大鼠(shu)用(yong)戊巴比妥钠腹腔注射麻醉，用(yong)干棉(mian)棒拭(shi)去结(jie)膜(mo)囊多(duo)余液(ye)(ye)体，将统一规格直径3mm的(de)圆(yuan)形滤(lv)纸片(pian)浸入1mol/LNaOH溶液(ye)(ye)中20s，饱和(he)后取出置于(yu)干燥滤(lv)纸上1秒以(yi)吸(xi)去过多(duo)的(de)碱液(ye)(ye)，将滤(lv)纸片(pian)贴(tie)敷于(yu)大鼠(shu)右眼(yan)角(jiao)膜(mo)**90s后移去，立即用(yong)灭(mie)菌(jun)水(shui)冲洗结(jie)膜(mo)囊及角(jiao)膜(mo)2min。2、检(jian)测标准：肉眼(yan)或裂隙(xi)灯(deng)下观(guan)察(cha)到角(jiao)膜(mo)有损(sun)伤。提供疾(ji)病动(dong)(dong)物(wu)(wu)模(mo)型(xing)(xing)建模(mo)公司通过小(xiao)鼠(shu)疾(ji)病模(mo)型(xing)(xing)的(de)构建有助于(yu)我们深入揭示潜在致(zhi)病基因(yin)作用(yong)机(ji)制。

本能发明(ming)的(de)(de)(de)(de)(de)(de)另(ling)一(yi)目的(de)(de)(de)(de)(de)(de)是(shi)公(gong)开了如(ru)(ru)上所述的(de)(de)(de)(de)(de)(de)方(fang)(fang)(fang)(fang)法(fa)(fa)(fa)(fa)制(zhi)备(bei)的(de)(de)(de)(de)(de)(de)鼠慢(man)(man)性背根(gen)神经(jing)(jing)压(ya)迫(po)(po)(po)(po)(po)(po)模(mo)型(xing)(xing)的(de)(de)(de)(de)(de)(de)用(yong)途是(shi)将(jiang)该模(mo)型(xing)(xing)用(yong)于腰(yao)(yao)(yao)椎(zhui)(zhui)间盘突(tu)出(chu)方(fang)(fang)(fang)(fang)法(fa)(fa)(fa)(fa)研(yan)(yan)(yan)究(jiu)和(he)/或(huo)影(ying)(ying)像(xiang)学(xue)检(jian)测(ce)研(yan)(yan)(yan)究(jiu)。进一(yi)步(bu)地，该模(mo)型(xing)(xing)用(yong)于与(yu)(yu)磁(ci)(ci)(ci)性相关的(de)(de)(de)(de)(de)(de)和(he)/或(huo)检(jian)测(ce)方(fang)(fang)(fang)(fang)面的(de)(de)(de)(de)(de)(de)研(yan)(yan)(yan)究(jiu)，比如(ru)(ru)经(jing)(jing)颅磁(ci)(ci)(ci)刺(ci)激(ji)和(he)核磁(ci)(ci)(ci)共(gong)振(zhen)。本发明(ming)利用(yong)压(ya)迫(po)(po)(po)(po)(po)(po)元件插入腰(yao)(yao)(yao)椎(zhui)(zhui)椎(zhui)(zhui)间孔，模(mo)拟腰(yao)(yao)(yao)椎(zhui)(zhui)间盘突(tu)出(chu)后突(tu)出(chu)物(wu)对神经(jing)(jing)根(gen)的(de)(de)(de)(de)(de)(de)持(chi)续(xu)(xu)慢(man)(man)性的(de)(de)(de)(de)(de)(de)机械压(ya)迫(po)(po)(po)(po)(po)(po)症状，有(you)(you)(you)益效果有(you)(you)(you)：1.应用(yong)机械压(ya)迫(po)(po)(po)(po)(po)(po)神经(jing)(jing)根(gen)模(mo)拟腰(yao)(yao)(yao)椎(zhui)(zhui)间盘突(tu)出(chu)病理，症状的(de)(de)(de)(de)(de)(de)病因学(xue)与(yu)(yu)临床情况接近；2.相关的(de)(de)(de)(de)(de)(de)疼痛行为类似于特定(ding)的(de)(de)(de)(de)(de)(de)慢(man)(man)性疼痛症状，且易于客观测(ce)量；3.制(zhi)备(bei)方(fang)(fang)(fang)(fang)法(fa)(fa)(fa)(fa)简(jian)单，对动(dong)物(wu)损伤小(xiao)，稳定(ding)性好，成功率高；4.造模(mo)关键(jian)材料(liao)压(ya)迫(po)(po)(po)(po)(po)(po)元件不(bu)受磁(ci)(ci)(ci)场干扰，无在磁(ci)(ci)(ci)场作(zuo)用(yong)下(xia)移位风险，有(you)(you)(you)利于后续(xu)(xu)对动(dong)物(wu)进行磁(ci)(ci)(ci)疗，经(jing)(jing)颅磁(ci)(ci)(ci)刺(ci)激(ji)等(deng)及核磁(ci)(ci)(ci)共(gong)振(zhen)等(deng)检(jian)查；5.造模(mo)关键(jian)材料(liao)压(ya)迫(po)(po)(po)(po)(po)(po)元件不(bu)影(ying)(ying)响磁(ci)(ci)(ci)场，不(bu)会对仪器和(he)核磁(ci)(ci)(ci)共(gong)振(zhen)等(deng)仪器产生不(bu)良影(ying)(ying)响；6.此(ci)方(fang)(fang)(fang)(fang)法(fa)(fa)(fa)(fa)获得的(de)(de)(de)(de)(de)(de)模(mo)型(xing)(xing)由(you)于采(cai)用(yong)了h62黄(huang)铜或(huo)聚乳酸材料(liao)制(zhi)备(bei)的(de)(de)(de)(de)(de)(de)压(ya)迫(po)(po)(po)(po)(po)(po)元件，使得研(yan)(yan)(yan)究(jiu)方(fang)(fang)(fang)(fang)法(fa)(fa)(fa)(fa)与(yu)(yu)检(jian)测(ce)方(fang)(fang)(fang)(fang)法(fa)(fa)(fa)(fa)不(bu)受磁(ci)(ci)(ci)场作(zuo)用(yong)限制(zhi)，丰(feng)富了腰(yao)(yao)(yao)椎(zhui)(zhui)间盘突(tu)出(chu)临床前研(yan)(yan)(yan)究(jiu)的(de)(de)(de)(de)(de)(de)方(fang)(fang)(fang)(fang)法(fa)(fa)(fa)(fa)及影(ying)(ying)像(xiang)学(xue)检(jian)测(ce)，为临床研(yan)(yan)(yan)究(jiu)提供(gong)理论依据。以下(xia)将(jiang)结(jie)合附图(tu)对本发明(ming)作(zuo)进一(yi)步(bu)说(shuo)(shuo)明(ming)，以充分说(shuo)(shuo)明(ming)本发明(ming)的(de)(de)(de)(de)(de)(de)目的(de)(de)(de)(de)(de)(de)、技术(shu)特征和(he)技术(shu)效果。附图(tu)说(shuo)(shuo)明(ming)图(tu)1是(shi)压(ya)迫(po)(po)(po)(po)(po)(po)元件插入椎(zhui)(zhui)间孔的(de)(de)(de)(de)(de)(de)结(jie)构示意图(tu)。

1、动(dong)(dong)物(wu)(wu)模(mo)(mo)型(xing)名(ming)称：皮(pi)肤(fu)浅II°及深(shen)II°烫伤(shang)(shang)(shang)大(da)(da)鼠模(mo)(mo)型(xing)2、实(shi)(shi)验(yan)(yan)(yan)(yan)动(dong)(dong)物(wu)(wu)种属：SD大(da)(da)鼠3、实(shi)(shi)验(yan)(yan)(yan)(yan)动(dong)(dong)物(wu)(wu)性别：雌雄不限4、实(shi)(shi)验(yan)(yan)(yan)(yan)动(dong)(dong)物(wu)(wu)年龄：成年5、实(shi)(shi)验(yan)(yan)(yan)(yan)动(dong)(dong)物(wu)(wu)体重：160-200g6、实(shi)(shi)验(yan)(yan)(yan)(yan)动(dong)(dong)物(wu)(wu)环境：SPF级1、实(shi)(shi)验(yan)(yan)(yan)(yan)方法：热力致皮(pi)肤(fu)损伤(shang)(shang)(shang)法。麻醉大(da)(da)鼠，根据体重腹(fu)部备皮(pi)，然后固定于(yu)(yu)(yu)实(shi)(shi)验(yan)(yan)(yan)(yan)台上。将(jiang)大(da)(da)鼠移近加热纯净水(shui)(shui)(shui)(shui)的(de)烧杯，将(jiang)纱(sha)(sha)布条浸(jin)入热水(shui)(shui)(shui)(shui)中(zhong)，待水(shui)(shui)(shui)(shui)温恒定于(yu)(yu)(yu)99℃时，取(qu)出纱(sha)(sha)布，立即平铺于(yu)(yu)(yu)待烫部位，于(yu)(yu)(yu)纱(sha)(sha)布接触大(da)(da)鼠皮(pi)肤(fu)后开始计(ji)时。致伤(shang)(shang)(shang)3s为浅II°烫伤(shang)(shang)(shang)，致伤(shang)(shang)(shang)10s为深(shen)II°烫伤(shang)(shang)(shang)。2、检测(ce)标准：a.皮(pi)肤(fu)大(da)(da)体观察：致伤(shang)(shang)(shang)3s大(da)(da)鼠局部皮(pi)肤(fu)发(fa)红、轻微(wei)水(shui)(shui)(shui)(shui)肿(zhong)。愈合(he)后毛发(fa)生长(zhang)良好(hao)，有少(shao)量红褐**素沉着，皮(pi)肤(fu)轻微(wei)挛缩，表皮(pi)光滑；致伤(shang)(shang)(shang)10s大(da)(da)鼠局部皮(pi)肤(fu)发(fa)白、水(shui)(shui)(shui)(shui)肿(zhong)。愈合(he)后毛发(fa)生长(zhang)良好(hao)，皮(pi)肤(fu)瘢痕形成，表面粗糙(cao)不平。构(gou)建人源移植瘤(liu)模(mo)(mo)型(xing)需要合(he)适的(de)免疫缺(que)陷小(xiao)鼠想知(zhi)道如(ru)何选(xuan)择(ze)？

因此(ci)，应用(yong)(yong)(yong)动物(wu)模(mo)(mo)型(xing)，除(chu)了能克(ke)服在(zai)人(ren)类(lei)研(yan)究(jiu)中(zhong)经常会(hui)遇(yu)到的(de)理论(lun)和社会(hui)限制(zhi)(zhi)外，还容许采用(yong)(yong)(yong)某些不能应用(yong)(yong)(yong)于人(ren)类(lei)的(de)方法学途(tu)径(jing)，甚至(zhi)为了研(yan)究(jiu)需要(yao)可(ke)(ke)以(yi)(yi)损伤动物(wu)组织、或处(chu)死动物(wu)。（二）临(lin)(lin)床上平时(shi)不易见到的(de)疾(ji)病(bing)(bing)(bing)(bing)(bing)可(ke)(ke)用(yong)(yong)(yong)动物(wu)随时(shi)复制(zhi)(zhi)出来(lai)临(lin)(lin)床上平时(shi)很难收集到放射病(bing)(bing)(bing)(bing)(bing)、毒(du)气中(zhong)毒(du)、烈性(xing)(xing)传(chuan)染病(bing)(bing)(bing)(bing)(bing)等病(bing)(bing)(bing)(bing)(bing)人(ren)，而(er)实验室可(ke)(ke)以(yi)(yi)根据研(yan)究(jiu)目的(de)要(yao)求随时(shi)采用(yong)(yong)(yong)实验性(xing)(xing)诱发(fa)(fa)的(de)方法在(zai)动物(wu)身(shen)(shen)上复制(zhi)(zhi)出来(lai)。（三）可(ke)(ke)以(yi)(yi)克(ke)服人(ren)类(lei)某些疾(ji)病(bing)(bing)(bing)(bing)(bing)潜伏(fu)期长(zhang)，病(bing)(bing)(bing)(bing)(bing)程长(zhang)和发(fa)(fa)病(bing)(bing)(bing)(bing)(bing)率低的(de)缺点一般遗传(chuan)性(xing)(xing)、免疫性(xing)(xing)、代(dai)谢(xie)性(xing)(xing)和内(nei)分泌(mi)等疾(ji)病(bing)(bing)(bing)(bing)(bing)在(zai)临(lin)(lin)床上发(fa)(fa)病(bing)(bing)(bing)(bing)(bing)率很低，例(li)如急(ji)性(xing)(xing)白(bai)血病(bing)(bing)(bing)(bing)(bing)的(de)发(fa)(fa)病(bing)(bing)(bing)(bing)(bing)率较降(jiang)，研(yan)究(jiu)人(ren)员可(ke)(ke)以(yi)(yi)有(you)意识(shi)地(di)提高其在(zai)动物(wu)种(zhong)群的(de)中(zhong)发(fa)(fa)生频率，从而(er)推进研(yan)究(jiu)。同(tong)样的(de)途(tu)径(jing)已成功(gong)地(di)应用(yong)(yong)(yong)于其他疾(ji)病(bing)(bing)(bing)(bing)(bing)的(de)研(yan)究(jiu)，如血友病(bing)(bing)(bing)(bing)(bing)、周期性(xing)(xing)中(zhong)性(xing)(xing)白(bai)细胞(bao)减少症和自身(shen)(shen)免疫介导(dao)性(xing)(xing)疾(ji)病(bing)(bing)(bing)(bing)(bing)等。动物(wu)疾(ji)病(bing)(bing)(bing)(bing)(bing)模(mo)(mo)型(xing)广(guang)泛应用(yong)(yong)(yong)于疾(ji)病(bing)(bing)(bing)(bing)(bing)机(ji)制(zhi)(zhi)研(yan)究(jiu)。徐(xu)州正规(gui)疾(ji)病(bing)(bing)(bing)(bing)(bing)动物(wu)模(mo)(mo)型(xing)建模(mo)(mo)

动物(wu)疾(ji)病模(mo)型(xing)的另一个富有成效的用途(tu)。徐州如何(he)使用疾(ji)病动物(wu)模(mo)型(xing)建模(mo)

转(zhuan)(zhuan)基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)小(xiao)(xiao)鼠对黑色素瘤(liu)发生(sheng)的分子途径(jing)的理解有(you)重(zhong)(zhong)要意(yi)义。此(ci)外，转(zhuan)(zhuan)基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)小(xiao)(xiao)鼠是(shi)(shi)可靠(kao)且可重(zhong)(zhong)现的模(mo)型(xing)(xing)(xing)，用(yong)来(lai)评估受(shou)损基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)对黑色素瘤(liu)生(sheng)物学的影响。应用(yong)：基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)工(gong)程小(xiao)(xiao)鼠模(mo)型(xing)(xing)(xing)可用(yong)于(yu)研(yan)(yan)究(jiu)特定(ding)基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)组改(gai)变在黑色素瘤(liu)的发生(sheng)和(he)发展(zhan)的重(zhong)(zhong)要性及药物的靶向。1Lum－/－基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)敲除小(xiao)(xiao)鼠Lumican是(shi)(shi)一种富(fu)含亮氨酸(suan)的小(xiao)(xiao)蛋白聚(ju)糖(smallleucine-richproteoglycan，SLRP)，为(wei)胶原原纤(xian)维(wei)形成(cheng)的关键调(diao)节(jie)剂。黑色素瘤(liu)中(zhong)Lumican表达降低(di)与(yu)(yu)浸润(run)相关。方(fang)法(fa)：有(you)研(yan)(yan)究(jiu)者(zhe)利用(yong)BamHI建(jian)立的克隆(long)衍生(sheng)物导(dao)入(ru)胚胎干细胞(bao)(bao)，建(jian)立Lum－/－转(zhuan)(zhuan)基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)小(xiao)(xiao)鼠。该(gai)模(mo)型(xing)(xing)(xing)可提供与(yu)(yu)发生(sheng)和(he)转(zhuan)(zhuan)移相关机制(zhi)及可见变的进展(zhan)，以(yi)(yi)及确定(ding)负责的黑色素瘤(liu)进展(zhan)的基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)。2Tyr::N-RasQ61K转(zhuan)(zhuan)基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)小(xiao)(xiao)鼠方(fang)法(fa)：有(you)研(yan)(yan)究(jiu)者(zhe)使用(yong)突变的人N-ＲasQ61K和(he)SV40剪接以(yi)(yi)及聚(ju)腺苷(gan)酸(suan)化序列生(sheng)成(cheng)Tyr::N-ＲasQ61K构建(jian)体，并(bing)注射到卵(luan)母细胞(bao)(bao)中(zhong)，建(jian)立Tyr::N-ＲasQ61K转(zhuan)(zhuan)基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)小(xiao)(xiao)鼠。3BrafV600E转(zhuan)(zhuan)基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)小(xiao)(xiao)鼠模(mo)型(xing)(xing)(xing)有(you)研(yan)(yan)究(jiu)者(zhe)使用(yong)LoxP-stop-LoxP(LSL)/Cre重(zhong)(zhong)组酶(mei)技术从内源性Braf基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)中(zhong)诱导(dao)BrafV600E表达，建(jian)立BrafV600E转(zhuan)(zhuan)基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)黑色素瘤(liu)小(xiao)(xiao)鼠模(mo)型(xing)(xing)(xing)。总结目前已有(you)三种不同的黑色素瘤(liu)小(xiao)(xiao)鼠模(mo)型(xing)(xing)(xing)，但由(you)于(yu)实(shi)验动物与(yu)(yu)人类基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)组、基(ji)(ji)(ji)(ji)(ji)(ji)(ji)因(yin)调(diao)控、细胞(bao)(bao)类型(xing)(xing)(xing)、组成(cheng)等方(fang)面是(shi)(shi)有(you)一定(ding)差别。徐(xu)州如何(he)使用(yong)疾病动物模(mo)型(xing)(xing)(xing)建(jian)模(mo)

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