条件性敲除软骨组织中FGF9基因小鼠模型的建立

目的：利用Cre-LoxP系统建立可调控的软骨组织条件性敲除成纤维细胞生长因子9（fibroblast growth factor,FGF9）小鼠模型。方法：构建针对小鼠Fgf9基因2号外显子的重组载体,电穿孔转染胚胎干细胞（ES细胞）。选择药物G418和Ganc,筛选阳性ES细胞并进行PCR鉴定。利用显微注射将ES细胞注入C57BL/6J小鼠囊胚,移入假孕小鼠子宫,获得含Neo的flox杂合小鼠。该小鼠去Neo后,与ColⅡ-Cre转基因小鼠杂交,并以他莫昔芬诱导,其后代软骨细胞内flox纯合、Cre阳性小鼠的flox区域被敲除。结果：Fgf9基因flox杂合子小鼠无明显异常,可稳定繁殖。该flox小鼠与ColⅡ-Cre转基因小鼠交配后,成功建立条件性敲除软骨组织中Fgf9基因小鼠模型。结论：利用Cre-LoxP系统,可成功建立Fgf9基因条件性敲除小鼠模型,为后续研究FGF9在骨软骨发育及骨关节稳态维持中的功能奠定了基础。     

诱导型成骨细胞特异Stat3基因敲除小鼠的构建及验证

目的：运用Cre-Loxp基因敲除系统,构建诱导型条件性Stat3基因敲除小鼠并验证其敲除效率。方法：通过Stat3^fl/fl与Col1 creERT2基因型的C57小鼠多代杂交,构建诱导型成骨细胞特异Stat3敲除小鼠Stat3^Col1ERT2。取其骨髓间充质干细胞（bone mesenchymal stem cells, BMSCs）,加入4-羟基他莫西芬（4-OTH）后,采用实时定量PCR技术和免疫印迹技术在mRNA与蛋白水平分别体外验证Stat3敲除效果。于小鼠腹腔注射他莫昔芬,采用免疫荧光染色技术,观察小鼠上颌牙槽骨区域STAT3的表达,以体内验证敲除效果。采用SPSS 24.0软件包对数据进行统计学分析。结果：实时定量PCR和免疫印迹结果显示,Stat3^Col1ERT2小鼠BMSCs体外加药诱导敲除后,STAT3的mRNA水平显著下调（P<0.05）、蛋白表达降低（P<0.05）。免疫荧光染色结果显示,Stat3^Col1ERT2小鼠体内上颌牙槽骨区域成骨细胞的STAT3表达显著降低（P<0.05）。结论：本研究成功构建了诱导型成骨细胞特异Stat3基因敲除小鼠,使基因敲除可受观察者时空调控,为今后正畸牙移动、颌骨牵引成骨、颌骨骨折等牙槽骨改建机制的研究提供了新的思路及依据。     

ALPL在BMMSCs调控血管内皮细胞管腔形成中的作用研究

目的:探究ALPL基因在骨髓间充质干细胞对内皮细胞(ECs)管腔形成及骨内H型血管的影响.方法:以正常C57BL/6小鼠骨髓间充质干细胞(mBMMSCs)及人骨髓间充质干细胞(hBMMSCs)为对照组,取ALPL+/-基因敲除小鼠mBMMSCs、HPP患者hBMMSCs及通过慢病毒浸染法敲减ALPL基因的hBMMSCs...     

GSK-3β对正畸牙齿移动的影响

目的:观察GSK-3β对正畸牙齿移动距离的影响.方法:分别选30 只野生C57小鼠和20 只GSK-3β基因敲除C57小鼠.野生型和基因敲除型各20 只,正畸加力3、5、7、14 d(5 只/组)后处死,取材固定上颌骨扫描Micro CT测量牙齿移动距离,冰冻切片免疫荧光染色观察破骨情况.野生型10 只腹腔注射LiCl(隔天100 ng/ml)加力3 d,7 d处死后扫描MicroCT.结果:与野生型组同期结果相比,GSK-3β基因敲除小鼠牙齿移动距离降低(P<0.05),压力侧破骨细胞减少(P<0.05);基因敲除组与野生型注射LiCl组牙齿移动距离无统计学差异.结论:GSK-3β可以影响破骨细胞形成从而影响正畸牙齿移动.     

腭裂相关基因甲状腺转录因子-2转基因小鼠的生物学特征

目的 建立腭裂相关基因甲状腺转录因子-2（TTF-2）转基因小鼠模型,利用转基因动物模型研究TTF-2基因的活动规律和表达模式发生变化时对腭突发育过程产生的影响。方法 采用聚合酶链反应法扩增C57BL/6J小鼠基因组 TTF-2基因,将其定向插入pBROAD3-mcs载体,构建重组表达质粒pBROAD3-TTF-2,利用显微注射技术把线性化表达载体注射到受精卵的雄原核中,建立TTF-2转基因小鼠。利用特异引物聚合酶链反应和Southern blot方法鉴定转基因小鼠的基因型,采用免疫组织化学法检测TTF-2基因在转基因小鼠腭突组织中的表达。结果 共注射原核期受精卵982枚,发育为2-细胞胚胎的580枚,均移植入48个假孕受体昆明白小鼠中,得到胎鼠 68只。提取基因组DNA,发现有13只转基因阳性的胎鼠。免疫组织化学法检测显示 TTF-2在转基因小鼠腭突中持续表达。结论 通过显微注射法使外源基因pBROAD3-TTF-2整合入小鼠基因组中,成功建立了腭突持续表达 TTF-2的转基因小鼠模型,其表现型为腭裂。     

长期低剂量牙龈卟啉单胞菌感染对脂蛋白基因敲除小鼠动脉粥样硬化形成的影响

目的 评价牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)口腔内低剂量、长期、多次接种是否可影响载脂蛋白基因敲除小鼠(apolipoprotein E-knocked out,ApoE-/-)的动脉粥样硬化(atherosclerosis,AS)形成进程和病变程度.方法 20只C57BL/6背景小鼠,将其中13只种属为C57BL/6背景的ApoE-/-小鼠按随机数字表随机分成两组,实验组7只使用1013个/L的牙周致病菌Pg进行口腔内涂菌接种,持续15周共75次,对照组6只同法口腔内涂擦无菌肉汤培养基,比较两组小鼠体质量、血总胆固醇、三酰甘油及主动脉AS病损面积及病理组织变化的情况.另7只野生型C57BL/6小鼠取其主动脉组织作为正常对照.结果 实验组ApoE-/-小鼠主动脉AS病损平均面积为(98 363.68±12 043.00)μm2,显著大于未接种的小鼠对照组[平均病损面积(62 985.06±7419.64)μm2],P=0.035;两组体质量、血总胆固醇和三酰甘油差异均无统计学意义;与野生型C57BL/6小鼠正常主动脉相比,ApoE-/-小鼠主动脉内壁均有凸向管腔内的AS病损,且动脉壁变平,实验组较对照组AS斑块更明显,动脉管腔更狭窄.结论 Pg口腔内低剂量长期多次接种可以加速ApoE-/-小鼠AS的进展.

Abstract：

Objective To assess the effect of longterm and lower oral inoculation with Porphyromonas gingivalis (Pg) on the progression of atherosclerosis in apolipoprotein E-knocked out (ApoE -/-) mice. Methods Six-week-old male ApoE -/- mice were inoculated orally with 0. 1 ml live Pg(1013/L) or bouillon culture-medium quintic per week for 15 consecutive weeks, altogether 75 times of inoculations. The lesion area of atherosclerosis in the aortic tree was measured by en face quantification by red oil O staining method. The atherosclerotic lesion was examined by histopathology. The levels of total cholesterol and triglycerides were compared. Results At 22 weeks after inoculation, the mean atherosclerotic lesion area in inoculated mice was (98 363.68 ± 12 043.00) μm2 ,which was significantly greater than that in noninoculated mice, which was (62 985.06 ± 7419. 64) μm2 (P = 0. 035).Conclusions Longterm lower oral inoculation of Pg can accelerate the progression of atherosclerosis in apolipoprotein E-knocked out mice.     

PTEN基因不同表达的黑色素瘤动物模型建立及鉴定

目的建立PTEN基因不同表达的黑色素瘤动物模型。方法 15只C57BL/6小鼠随机分为三组,每组5只。对照组:注入0.2ml RPMI-1640培养液;实验组A:注入0.2ml浓度为4×10^6/ml的(PTEN+/+-B16F10);实验组B:注入0.2ml浓度为4×10^6/ml的(PTEN-/--B16F10)。实验组中80%小鼠精神萎靡时停止实验。采用免疫印迹检测肿瘤细胞和成瘤组织中PTEN蛋白表达情况。结果 A、B两组小鼠成瘤率100%。A、B两组小鼠肿瘤体积比较有统计学差异(P<0.05)。免疫印迹检测细胞(PTEN+/+-B16F10)与A组小鼠成瘤组织中PTEN蛋白表达呈阳性;细胞(PTEN-/--B16F10)与B组小鼠成瘤组织中PTEN蛋白表达呈阴性。结论成功构建了PTEN基因不同表达的黑色素瘤小鼠模型;PTEN基因与黑色素瘤的生长有相关性。     

条件性血管瘤转基因小鼠模型的建立

目的：采用条件性转基因策略构建小鼠血管瘤动物模型,并对其表型进行鉴定。方法：构建血管内皮细胞特异性表达启动子Tie2驱动的鼠多瘤病毒中间T基因（ PyMT）表达质粒（ pTie2?PyMT）,采用DNA显微注射方法,将血管内皮特异性表达的PyMT目的基因导入供体C57BL／6J小鼠的雄原核中,再移植到假孕鼠的输卵管中,产生转基因首建鼠。 PCR方法检测目的基因整合情况,检查转基因鼠基因型,观察转基因鼠表型,对于转基因鼠出现的新生物进行组织学及免疫组织化学检测。采用GraphPad Prism 5．0软件包对实验数据进行统计学分析。结果：经测序分析证实,pTie2?PyMT质粒中PyMT、Tie2启动子和Tie2增强子序列等组成元件被正确克隆、连接,且阅读框正确。出生小鼠基因型经PCR鉴定证实,阳性的转基因小鼠均出现血管瘤样新生物表型。血管瘤样新生物主要表达部位在小鼠的耳、舌、皮肤、黏膜、肝等部位,组织学检查证实为血管瘤样病变,免疫组织化学方法证实新生物的内皮细胞表达PyMT蛋白及血管内皮标志物CD31。结论：Tie2启动子驱动下的PyMT基因可以在小鼠体内诱发血管瘤,该模型鼠可用于血管瘤发病机制的研究。条件控制下的转基因技术是一种有效的建立血管瘤动物模型的方法。     

Myo1h基因敲除小鼠模型的建立与初步观察

目的 构建Myo1h(Myosin 1H)基因与人同源的第30号外显子敲除模型小鼠(Myo1h小鼠)并验证其敲除效率。方法 采用Crispr/Cas9技术构建Myo1h基因敲除F0(the founder)代嵌合体小鼠,并进行配笼繁殖,经过基因型鉴定后获得纯合F2(the second filial generation,子2代)代实验鼠。利用实时荧光定量PCR技术和免疫印迹技术从mRNA水平和蛋白层面验证敲除效果;利用免疫荧光染色技术,对小鼠髁突区域Myo1h的表达进行观察,从体内验证敲除效果;通过Micro-CT三维重建测量有效下颌骨长度,对F2代小鼠进行表型评估;采用Prism 8.0软件包对数据进行统计学分析。结果 实时定量PCR和免疫印迹结果显示,Myo1h基因的mRNA水平显著降低(P<0.05)、蛋白表达降低。免疫荧光染色结果显示,Myo1h小鼠体内脑部、肺部以及髁突组织的Myo1h表达显著降低(P<0.05)。通过Micro-CT和身长测量分析进行表型评估后发现,Myo1h基因敲除后小鼠的有效身长比正常对照组短(P<0.05)。结论 本研究成功构建出M...     

主穹隆蛋白通过抑制破骨细胞分化在牙周炎中起骨保护作用

目的:通过建立小鼠实验性牙周炎模型及体外骨髓间充质干细胞(BMMSCs)破骨细胞向诱导,探讨主穹隆蛋白(MVP)在牙周炎骨吸收中的作用。方法:MVP基因敲除(MVP-/-)和野生型(WT)C57BL/6小鼠分别局部注射脂多糖(LPS)以建立实验性牙周炎模型,通过micro CT扫描、耐酒石酸酸性磷酸酶(TRAP)染色等方法检测骨吸收程度。同时,体外分离培养MVP-/-与WT C57BL/6小鼠的BMMSCs,并诱导其向破骨细胞分化,通过TRAP染色、麦胚凝集素(WGA)染色等方法观察MVP对BMMSCs破骨向分化及骨吸收活性的影响。结果:在LPS诱导的小鼠实验性牙周炎中,MVP-/-组小鼠牙周炎骨吸收更为明显,且在注射区域内可见更多破骨细胞;体外实验证明,MVP-/-组小鼠的BMMSCs分化形成更多的破骨细胞,且骨吸收更明显。结论:MVP可以抑制破骨细胞分化,在牙周炎中起骨保护作用。     

Effects of Toll-like receptor 4 on Porphyromonas gingivalis-induced bone loss in mice

Background and Objective:  Toll-like receptor 4 (TLR-4)/myeloid differentiation protein-2 complex ligation by lipopolysaccharide induces production of pro-inflammatory cytokines and co-stimulatory molecules on antigen presenting cells. The aim of this study was to determine the role of the TLR-4 in bone loss-resistant C57BL mice and in bone loss-susceptible BALB/c mice after infection with Porphyromonas gingivalis .
Material and Methods:  The BALB/c and C57BL/10 mice, either normal or TLR-4 deficient, were infected or sham-infected orally four times, at 4 day intervals, with 10⁹ colony forming units of P. gingivalis . At 47 days, defleshed jaws were stained and photographed in a standardized position. We measured the surface area of the root trunk to assess the alveolar bone loss.
Results:  Porphyromonas gingivalis -infected wild-type BALB/c mice lost 13.8% more bone than P. gingivalis -infected wild-type C57BL/10 mice. In contrast, P. gingivalis -infected TLR-4-deficient C57BL/10 mice lost 12.7% more bone than P. gingivalis -infected TLR-4-deficient BALB/c mice. Porphyromonas gingivalis -infected wild-type C57BL/6 and TLR-2 knockout C57BL/6 mice had similar bone levels to sham-infected control mice.
Conclusion:  Toll-like receptor 4 is protective for C57BL/10 but detrimental to BALB/c mice, since its absence allowed C57BL/10 but not BALB/c mice to lose alveolar bone. Toll-like receptor 2 does not contribute to this protection in genetically similar C57BL/6 mice.     

Caries susceptibility in inbred mouse strains and inheritance patterns in F1 and backcross (N2) progeny from strains with high and low caries susceptibility.

We studied dental caries susceptibility in various inbred mice strains infected with Streptococcus mutans and the inheritance pattern in the F1 and the N2 backcross animals. A high caries score was observed in four laboratory strains, BALB/cAJcl, C57BL/6NJcl, C57BL/10Slc and DBA/2NJcl. Three strains, C3H/HeNJcl, AKR/JSlc and CBA/JNCrj, showed less caries. Males of strain C57BL/10Slc (mean caries score = 112.2) and females of strain C3H/HeNJcl (mean caries score = 24.0) were chosen for examinations of the inheritance of the caries susceptibility. The mean caries score in (C57BL/10Slc x C3H/HeNJcl) F1 hybrids was 98.4, demonstrating that F1 progenies were susceptible. A number of N2 mice were obtained by mating the F1 male and the C3H/HeNJcl female. The caries scores of these N2 male mice had an extensive range, from 14 to 194. Assuming that a caries score over 74 (median of the scores between C57BL/10Slc and C3H/HeNJcl) belonged to the highly caries-susceptible group, N2 mice could be divided into groups with low or high caries susceptibility. Furthermore, the effect of nu/nu mutation on caries susceptibility in mice was also examined.     

不同母体环境下小鼠腭胚突Fgf10/Fgfr2b/Shh信号通路的变化

目的：观察在腭发育关键时期不同母体环境对小鼠腭胚突Fgf10信号表达的影响。方法：建立A/WySnJ/C57BL/6两系小鼠胚胎移植模型,利用RT-PCR技术检测在胚胎12.5和13.5d胎鼠腭突Fgf10/Fgfr2b/Shh的表达。结果：胚胎12.5d,A/C两系小鼠受精卵相互之间移植前后比较,胎鼠腭突Fgf10和Fgfr2b的表达没有明显变化,A系小鼠移植到C系小鼠后,胎鼠腭突Shh表达显著下调（P〈0.01）;胚胎13.5d,A系小鼠移植到C系小鼠后,胎鼠腭突Fgf10和Fgfr2b的表达显著上调（P〈0.01）,Shh的表达显著下调（P〈0.01）,相反,C系小鼠移植到A/系小鼠后,胎鼠腭突Fgf10的水平显著下调,而Shh水平显著上调（P〈0.01）。结论：母体子宫微环境和对外界环境反应的改变可影响胚胎腭突发育过程中关键信号因子的表达。     

Mapping of a gene causing mouse gutter-shaped tooth root to chromosome 5

A study was conducted to identify the major candidate chromosome and to detect the region which included the candidate gene causing gutter-shaped root (GSR) in an inbred strain of mice. The candidate chromosomal analysis was performed on genetic crosses of C57L/J strain mice, which have GSR, and C57BL/6J strain mice, which have normal roots. Linkage analysis suggested that mouse chromosome 5 was one of the major candidates and therefore this chromosome was investigated in detail by individual genotyping of all backcross mice. The highest linkage was found at D5Mit161, and other high linkages were evident at D5Mit29, 321 and 427. Based on these findings, it is suggested that the gene causing GSR formation in mice maps close to these microsatellite loci.     

Early activation of the interleukin-23-17 axis in a murine model of oropharyngeal candidiasis

Candida albicans is an oral commensal yeast that causes oropharyngeal candidiasis (OPC) in immunocompromised individuals. The immunological pathways involved in OPC have been revisited after the interleukin-17 (IL-17) pathway was implicated in fungal immunity. We studied immediate (<24 h) and adaptive (3-6 day) IL-12 and IL-23-17 pathway activation in naive p40(-/-) mice, which lack IL-12 and IL-23 and develop severe, chronic OPC upon oral inoculation with C. albicans. Macrophages from p40(-/-) mice were less efficient than C57BL/6J controls at killing C. albicans in vitro but very low numbers in the oral mucosae of infected C57BL/6J mice suggest that they are not critical in vivo, at least in this strain. Migration of macrophages to regional lymph nodes of infected p40(-/-) mice was impaired; however, dendritic cell migration was not affected. Recombinant IL-12 therapy provided only temporary relief from OPC, suggesting that IL-23 is required for full protection. In C57BL/6J mice, but not p40(-/-) mice, messenger RNAs encoding IL-23p19 and IL-17 were induced in the oral mucosa within 24 h of infection (6 ± 0.6 and 12 ± 2.7-fold). By day 6 of infection in C57BL/6J mice, IL-17A messenger RNA level had increased 5.1 ± 1.8 and 83 ± 21-fold in regional lymph nodes and oral tissues respectively. Ablation of p40 was associated with delayed or abrogated induction of IL-17A pathway targets (monocyte chemoattractant protein-1, IL-6 and macrophage inflammatory protein-2), and a lack of organized recruitment of neutrophils to the infected oral mucosa. Overall our data show that the IL-23-17A axis is activated early in the oral mucosae of immunologically naive mice with OPC.     

Susceptibility of type 2 diabetic mice to low-virulence bacterial infection: induction of abscess formation by gingipain-deficient Porphyromonas gingivalis

BACKGROUND AND OBJECTIVE: Type 2 diabetes mellitus is considered an important risk factor of adult periodontitis. However, recent studies have revealed that the subgingival microbial flora of diabetes mellitus patients does not differ from that of healthy individuals. In this study, we examined the response of type 2 diabetes mellitus hosts to low-virulence bacteria in a murine abscess model. MATERIAL AND METHODS: Porphyromonas gingivalis ATCC 33277 or KDP128 (rgpA rgpB kgp) were injected into two mouse strains - C57BL/6J and its derivative, KK/A(Y), which becomes diabetic spontaneously. RESULTS: Lesions of KK/A(Y) mice injected with either low-virulence P. gingivalis KDP128 or wild-type 33277 were significantly larger than those of C57BL/6J mice injected with the same strains. Histologically, more neutrophils and macrophages migrated to the lesions in the KK/A(Y) mice injected with P. gingivalis 33277 and KDP128 compared with those of C57BL/6J mice injected with the same respective strains. CONCLUSION: These results suggest that severe inflammation is observed in response to low-virulence bacteria in addition to the highly virulent bacteria in type 2 diabetes mellitus hosts.     

Susceptibility of different mouse strains to peri‐implantitis

Background and Objective

Peri‐implantitis (PI) is an inflammatory condition that affects the tissues surrounding dental implants. Although the pathogenesis of PI is not fully understood, evidence suggests that the etiology is multifactorial and may include a genetic component. The aim of this study was to investigate the role of genetics in the development of peri‐implantitis.

Material and Methods

Four‐week‐old C57BL/6J, C3H/HeJ and A/J male mice had their left maxillary molars extracted. Implants were placed in the healed extraction sockets. Upon osseointegration, ligatures were placed around the implant head for 1 or 4 weeks to induce PI. Micro‐computed tomography scanning was used to measure volumetric bone loss. Histological analyses were also performed to evaluate collagen organization and the presence of neutrophils and osteoclasts.

Results

Radiographically, comparing the ligature‐treated mice, C57BL/6J displayed the greatest amount of bone loss, followed by C3H/HeJ and A/J mice at 1 and 4 weeks. Histologically, at 1 week, C57BL/6J mice presented with the highest numbers of neutrophils and osteoclasts. At 4 weeks, C57BL/6J mice presented with the most active bone remodeling compared with the other two strains.

Conclusion

There were significant differences in the severity of peri‐implantitis among the different mouse strains, suggesting that the genetic framework can affect implant survival and success. Future work is needed to dissect the genetic contribution to the development of peri‐implantitis.     

The influence of genetic variation on the splenic T cell cytokine and specific serum antibody responses to Porphyromonas gingivalis in mice

BACKGROUND: T cell cytokine profiles in the spleens and anti-Porphyromonas gingivalis antibodies in the sera of P. gingivalis-immunized BALB/c (H-2d), CBA/CaH (H-2k), C57BL6 (H-2b), and DBA/2J (H-2d, C5 deficient) mice were examined. METHODS: Mice were immunized either by intraperitoneal injections of P. gingivalis outer membrane antigens and Freund's incomplete adjuvant weekly for 3 weeks or sham-immunized with PBS and adjuvant, followed by subcutaneous challenge with live organisms 1 week after the final immunization. Spleens were excised and blood samples collected by heart puncture at 0 and 7 days after challenge. Splenic CD4 and CD8 cells were stained for intracytoplasmic interleukin (IL)-4, interferon (IF)-gamma, and IL-10 and levels of anti-P. gingivalis antibodies in the serum samples determined by ELISA. RESULTS: Lesion sizes in immunized BALB/c mice remained stable for the 7-day experimental period. Immunized CBA/CaH and C57BL6 mice exhibited large lesions at day 1 reducing by day 7 particularly in the latter strain. Lesions in immunized DBA/2J mice were still larger than the other strains at day 7. With the exception of DBA/2J mice, sham-immunized mice demonstrated lesions which did not show signs of healing by day 7. T cell cytokine responses in sham-immunized mice at day 0 were low, increasing to a variable degree by day 7 after challenge in the 4 strains. Immunized BALB/c mice demonstrated intermediate T cell responses while generally exhibiting a stronger IFN-gamma response than IL-4 or IL-10. Immunized CBA/CaH and C57BL6 mice showed weak T cell cytokine responses while immunized DBA/2J displayed the strongest T cell responses particularly in regard to IL-4 positive cells. Sham-immunized mice had low levels of serum anti-P. gingivalis antibody levels at day 0 with levels increasing significantly by day 7 after challenge. Antibody levels in immunized mice seemed to correlate with lesion sizes. Immunized C57BL6 mice had the highest antibody levels followed by CBA/CaH, BALB/c with DBA/2J exhibiting low levels. The T cell and B cell antibody responses in each strain appeared to exhibit an inverse relationship. CONCLUSIONS: This study has shown that genetic differences at the level of H-2 haplotype induce variations in the local and T and B cell responses to P. gingivalis antigens. The responses of DBA/2J mice which have the same haplotype as BALB/c mice suggest that factors other than H-2 haplotype such as the C5 deficiency may influence this immune response. The significance of the specific antibody and T cell responses and of their inverse relationship to susceptibility to periodontal disease remains to be determined.