черновик. ЧЕРНОВИК. Применение инбредных мышей линий balbc, Nude, C57BL6 в качестве экспериментальных моделей.

• Название: Применение инбредных мышей линий balbc, Nude, C57BL6 в качестве экспериментальных моделей.
• Анкор: черновик
• Дата: 20.02.2023
• Размер: 77.45 Kb.
• Имя файла: ЧЕРНОВИК.docx
• Тип: Курсовая; #947389
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федеральное государственное образовательное учреждение высшего

образования «Тюменский государственный медицинский университет»

Министерства здравоохранения Российской Федерации

(ФГБОУ ВО «Тюменский ГМУ» Минздрава России»)

Кафедра гистологии с эмбриологией
Заведующий кафедрой:

Д.м.н., профессор Соловьев Г.С.


Курсовая работа

На тему : «Применение инбредных мышей линий BALB/c, Nude, C57BL/6 в качестве

экспериментальных моделей.»

Выполнил:

Студент: 2 курса 209 группы

Лаврентьева Юлия Ивановна

______ (подпись)

Руководитель:

Профессор, к.м.н.

Шидин Владимир Александрович

________________ (подпись)


Тюмень, 2021

Введение.

Существует множество видов экспериментальных животных моделей. Особое место среди них занимают мыши с нокаутированным геном (какой-либо ген либо

удален из ДНК или «выключен» для получения определенного эффекта). Моя задача в данной работе была изучить вопрос применения BALB/c, Nude, C57BL/6 видов мышиных линий в различных экспериментах и оценить их эффективность.

Инбредные животные - это животные, полученные путем инбридинга (in breed - выводить породу, разводить), т.е. последовательных близкородственных скрещиваний с целью получения гомозиготного и генетически идентичного потомства. Каждый инбредный штамм представляет собой уникальное сочетание генетического материала, порождающее уникальный фенотип. Многие такие фенотипные черты полезны в исследованиях, некоторые позволяют вывести «модели» болезней, прочие дают полезные физиологические, анатомические или поведенческие характеристики. Эти

индивидуальности, хорошо отраженные в литературе, представляют одну из

самых полезных инбредных черт. Для проведения фундаментальных исследований в иммунологии лучший объект - инбредные мыши.

Мыши инбредной линии BALB/c чаще других используются в экспериментах. Это одна из наиболее старых линий. Основателем этой линии является американский зоолог Хелси Багг, который получив несколько белых коммерческих мышей из Огайо, разводил их как закрытую колонию и создал линию Bagg Albino C. Эта линия с 1932 года известна под названием BALB/c. Она считается нераковой, хотя частота опухолей молочных желёз, лёгких и почек составляет в некоторых сублиниях 20-40%. Самки BALB/c не имеют фактора молока, их используют как кормилиц. Мыши этой линии подвержены заболеваниям сердечно-сосудистой системы и отличаются высоким систолическим давлением крови. Они чувствительны к желудочно-кишечным инфекциям. Их используют в разработке новых биомедицинских технологий.

Nude (лишенные волосяного покрова) - мыши со спонтанной мутацией, в результате которой у мышей-гомозигот по данной мутации (nu/nu) отсутствуют тимус и волосяной покров. Мутантный ген поддерживают при размножении мышей в гетерозиготном состоянии; он перенесен мышам нескольких других линий, например Balb/c, C57BL/6. Мыши линии Nude менее всех других подходят для этой цели вследствие своего неполного

иммунодефицита: они атимичны, и сниженное производство Т-клеток подавляет адаптивный ответ, однако неповрежденный врожденный иммунитет значительно ограничивает их полезность при трансплантации опухоли человека, особенно в случаях менее агрессивных, медленно растущих типах злокачественных новообразований.

Мыши линии С57BL/6 используются как физиологические или патологические модели для экспериментов in vivo, в качестве основной линии для генерации спонтанных и индуцированных мутаций, для создания трансгенных моделей мышей, а также при изучении заболеваний сердечно-сосудистой системы и эндокринных заболеваний. Животные данной линии хорошо размножаются, являются долгоживущими и имеют низкую частоту возникновения опухолей. Гемопоэтические стволовые клетки, полученные от мышей C57BL/6, демонстрируют сильно замедленное старение по сравнению с BALB/c.

Таким образом, правильный выбор вида и линии экспериментального животного и создание адекватной модели увеличивает шансы на то, что исследуемое

вещество будет успешным на этапе клинических испытаний.


1. Dong, J. Incubation and application of transgenic green fluorescent nude mice in visualization studies on glioma tissue remodeling / J. Dong, X.L. Dai, Z.H. Lu, X.F. Fei, H. Chen, Q.B. Zhang, Y.D. Zhao, Z.M. Wang, A.D. Wang, Q. Lan, Q. Huang // Chin Med J (Engl), 2012. – V. 125. – I. 24. – P. 4349-54.
Abstract

Background: The primary reasons for local recurrence and therapeutic failure in the treatment of malignant gliomas are the invasion and interactions of tumor cells with surrounding normal brain cells. However, these tumor cells are hard to be visualized directly in histopathological preparations, or in experimental glioma models. Therefore, we developed an experimental human dual-color in vivo glioma model, which made tracking solitary invasive glioma cells possible, for the purpose of visualizing the interactions between red fluorescence labeled human glioma cells and host brain cells. This may offer references for further studying the roles of tumor microenvironment during glioma tissue remodeling.
Methods: Transgenic female C57BL/6 mice expressing enhanced green fluorescent protein (EGFP) were crossed with male Balb/c nude mice. Then sib mating was allowed to occur continuously in order to establish an inbred nude mice strain with 50% of their offspring that are EGFP positive. Human glioma cell lines U87-MG and SU3 were transfected with red fluorescent protein (RFP) gene, and a rat C6 glioma cell line was stained directly with CM-DiI, to establish three glioma cell lines emitting red fluorescence (SU3-RFP, U87-RFP, and C6-CM-DiI). Red fluorescence tumor cells were inoculated via intra-cerebral injection into caudate nucleus of the EGFP nude mice. Tumor-bearing mice were sacrificed when their clinical symptoms appeared, and the whole brain was harvested and snap frozen for further analysis. Confocal laser scanning microscopy was performed to monitor the mutual interactions between tumor cells and host brain cells.
Results: Almost all the essential tissues of the established EGFP athymic Balb/c nude mice, except hair and erythrocytes, fluoresced green under excitation using a blue light-emitting flashlight with a central peak of 470 nm, approximately 50% of the offsprings were nu/nu EGFP+. SU3-RFP, U87-RFP, and C6-CM-DiI almost 100% expressed red fluorescence under the fluorescence microscope. Under fluorescence microscopic view, RFP+ cells were observed growing wherever they arrived at, locating in the brain parenchyma, ventricles, and para-vascular region. The interactions between the transplanted tumor cells and host adjacent cells could be classified into three types: (1) interweaving; (2) mergence; and (3) fusion. Interweaving was observed in the early stage of tumor remodeling, in which both transplantable tumor cells and host cells were observed scattered in the tumor invading and spreading area without organic connections. Mergence was defined as mutual interactions between tumor cells and host stroma during tumorigenesis. Direct cell fusion between transplantable tumor cells and host cells could be observed occasionally.
Conclusions: This study showed that self-established EGFP athymic nude mice offered the possibility of visualizing tumorigenesis of human xenograft tumor, and the dual-color xenograft glioma model was of considerable utility in studying the process of tumor remodeling. Based on this platform, mutual interactions between glioma cells and host tissues could be observed directly to further elucidate the development of tumor microenvironment.
2. Watts, C.J. Resistance of athymic nude mice to experimental cutaneous Bacillus anthracis infection / C.J. Watts, B.L. Hahn, P.G. Sohnle // J Infect Dis, 2009. – V. 199. – I. 5. -P. 673-679.

Abstract

Background: Previous studies in a murine cutaneous anthrax model have demonstrated that hairless and haired HRS/J mice are extremely resistant to Bacillus anthracis. Because these mice are relatively thymus deficient, we used C57BL/6 athymic nude and euthymic mice to evaluate the relationship between T cell deficiency and this heightened resistance.
Methods: Animals were epicutaneously inoculated with 1 × 107 B. anthracis (Sterne) spores onto abraded skin or injected with the spores intradermally or subcutaneously. The mice were then either monitored for survival or killed for quantitative histological experiments.
Results: Athymic mice were found to be markedly resistant to all 3 inoculation routes, compared with euthymic C57BL/6 mice. Athymic mice rendered leukopenic with cyclophosphamide became susceptible. Histological examination demonstrated increased inflammation and absence of organisms in the skin of athymic mice, compared with euthymic ones. The numbers of organisms in the athymic animals increased markedly after cyclophosphamide treatment. Superficial exudate fluids of inoculated skin showed many more neutrophils and ingested bacilli in the athymic mice.
Conclusions: These experiments demonstrate that athymic nude C57BL/6 mice are markedly resistant to experimental cutaneous anthrax, apparently because of a superficial neutrophilic response that clears the inoculated organisms before they can invade the underlying skin.
3. Dandekar, A.A. Virus-induced demyelination in nude mice is mediated by gamma delta T cells / A.A. Dandekar, S. Perlman // Am J Pathol, 2002. - V. 161. – I. 4. – P. 1255-1263.
Abstract

Infection of mice with mouse hepatitis virus (MHV), strain JHM, results in acute and chronic demyelination with many similarities to the human disease multiple sclerosis. This pathological process is primarily T cell-mediated and MHV infection of mice lacking B and T cells does not result in demyelination. In apparent contradiction to these results, robust demyelination is detected in MHV-infected young nude (athymic) mice. Herein, we show that demyelination in nude mice was mediated by γδ T cells. These cells, but not conventional CD4 or CD8 αβ T cells, were detected in the central nervous system of MHV-infected nude mice and their depletion with neutralizing antibody resulted in an 80% reduction in demyelination. These results show, for the first time, that γδ T cells can substitute for αβ T cells in a virus model of demyelination and further support a pathological role for γδ T cells in patients with multiple sclerosis.
4. Stemmer, K. Thermoneutral housing is a critical factor for immune function and diet-induced obesity in C57BL/6 nude mice / P. Kotzbeck, F. Zani, et al. // Int J Obes (Lond), 2015. – V.39 – I. 5. – P. 791-797.
Abstract

OBJECTIVES : Obesity-related cancers represent public health burdens of the first order. Nevertheless, suitable mouse models to unravel molecular mechanisms linking obesity to human cancer are still not available. One translational model is the immunocompromised Foxn1 (winged-helix/forkead transcription factor) nude mouse transplanted with human tumor xenografts. However, most xenograft studies are conducted in nude mice on an in-bred BALB/c background that entails protection from diet-induced obesity. To overcome such resistance to obesity and its sequelae, we here propose the dual strategy of utilizing Foxn1 nude mice on a C57BL/6 background and housing them at their thermoneutral zone.
METHODS: C57BL/6 nude and corresponding wild-type mice, housed at 23 or 33 °C, were subjected to either low-fat diet or high-fat diet (HFD). Energy expenditure, locomotor activity, body core temperature, respiratory quotient as well as food and water intake were analyzed using indirect calorimetry. Immune function at different housing temperatures was assessed by using an in vivo cytokine capture assay.
RESULTS: Our data clearly demonstrate that conventional housing protects C57BL/6 nude mice from HFD-induced obesity, potentially via increased energy expenditure. In contrast, HFD-fed C57BL/6 nude mice housed at thermoneutral conditions develop adiposity, increased hepatic triglyceride accumulation, adipose tissue inflammation and glucose intolerance. Moreover, increased circulating levels of lipopolysaccharide-driven cytokines suggest a greatly enhanced immune response in C57BL/6 nude mice housed at thermoneutrality.
CONCLUSION: Our data reveals mild cold stress as a major modulator for energy and body weight homeostasis as well as immune function in C57BL/6 nude mice. Adjusting housing temperatures to the thermoneutral zone may ultimately be key to successfully study growth and progression of human tumors in a diet-induced obese environment.
5. Wang, J.W. Immunologic Control of Mus musculus Papillomavirus Type 1 / J.W. Wang, R. Jiang, S. Peng, Y.N. Chang, C.F. Hung, R.B. Roden // PLoS Pathog. - 2015. – V. 11. – I. 10: e1005243. - DOI: 10.1371/journal.ppat.1005243

Abstract

Persistent papillomas developed in 10% of out-bred immune-competent SKH-1 mice following MusPV1 challenge of their tail, and in a similar fraction the papillomas were transient, suggesting potential as a model. However, papillomas only occurred in BALB/c or C57BL/6 mice depleted of T cells with anti-CD3 antibody, and they completely regressed within 8 weeks after depletion was stopped. Neither CD4+ nor CD8+ T cell depletion alone in BALB/c or C57BL/6 mice was sufficient to permit visible papilloma formation. However, low levels of MusPV1 were sporadically detected by either genomic DNA-specific PCR analysis of local skin swabs or in situ hybridization of the challenge site with an E6/E7 probe. After switching to CD3+ T cell depletion, papillomas appeared upon 14/15 of mice that had been CD4+ T cell depleted throughout the challenge phase, 1/15 of CD8+ T cell depleted mice, and none in mice without any prior T cell depletion. Both control animals and those depleted with CD8-specific antibody generated MusPV1 L1 capsid-specific antibodies, but not those depleted with CD4-specific antibody prior to T cell depletion with CD3 antibody. Thus, normal BALB/c or C57BL/6 mice eliminate the challenge dose, whereas infection is suppressed but not completely cleared if their CD4 or CD8 T cells are depleted, and recrudescence of MusPV1 is much greater in the former following treatment with CD3 antibody, possibly reflecting their failure to generate capsid antibody. Systemic vaccination of C57BL/6 mice with DNA vectors expressing MusPV1 E6 or E7 fused to calreticulin elicits potent CD8 T cell responses and these immunodominant CD8 T cell epitopes were mapped. Adoptive transfer of a MusPV1 E6-specific CD8+ T cell line controlled established MusPV1 infection and papilloma in RAG1-knockout mice. These findings suggest the potential of immunotherapy for HPV-related disease and the importance of host immunogenetics in the outcome of infection.
6. Kober, C. Intratumoral INF-γ triggers an antiviral state in GL261 tumor cells: a major hurdle to overcome for oncolytic vaccinia virus therapy of cancer / C. Kober, S. Weibel, S. Rohn, L. Kirscher, A.A. Szalay // Mol Ther Oncolytics. - 2015. – V. 2. - P. 15009 – DOI: 10.1038/mto.2015.9

Abstract

Oncolytic vaccinia virus (VACV) therapy is an alternative treatment option for glioblastoma multiforme. Here, we used a comparison of different tumor locations and different immunologic and genetic backgrounds to determine the replication efficacy and oncolytic potential of the VACV LIVP 1.1.1, an attenuated wild-type isolate of the Lister strain, in murine GL261 glioma models. With this approach, we expected to identify microenvironmental factors, which may be decisive for failure or success of oncolytic VACV therapy. We found that GL261 glioma cells implanted subcutaneously or orthotopically into Balb/c athymic, C57BL/6 athymic, or C57BL/6 wild-type mice formed individual tumors that respond to oncolytic VACV therapy with different outcomes. Surprisingly, only Balb/c athymic mice with subcutaneous tumors supported viral replication. We identified intratumoral IFN-γ expression levels that upregulate MHCII expression on GL261 cells in C57BL/6 wild-type mice associated with a non-permissive status of the tumor cells. Moreover, this IFN-γ-induced tumor cell phenotype was reversible.
7. Abe, Y. Characteristics of viruses derived from nude mice with persistent measles virus infection / K. Hashimoto, M. Watanabe, et al. // J Virol, 2013. – V. 87. – I. 8. – P. 4170-4175.
ABSTRACT

Measles virus (MV) isolates from patients with subacute sclerosing panencephalitis (SSPE) differ from wild-type MV virologically. However, few animal models have reported viruses with characteristics of the SSPE virus. The MV Edmonston strain was inoculated into the subarachnoid space of nude mice. All nude mice displayed weight loss and required euthanasia, with a mean survival duration of 73.2 days. The viral load in the brain was 4- to 400-fold higher than the inoculated load, and brain infection was confirmed by immunostaining. Gene sequencing of the viruses revealed that amino acid mutations occurred more frequently in matrix proteins. The most common mutation was a uridine-to-cytosine transition. The virus exhibited lower free virus particle formation ability than the Edmonston strain. When nude mice were challenged with 2 × 102 PFU of the brain-derived virus, the mean survival duration was 34.7 days, which was significantly shorter than that of the mice challenged with 4 × 104 PFU of the Edmonston strain (P < 0.01). This study indicated that MV in a nude mouse model of persistent infection exhibited characteristics of the SSPE virus. This model may prove useful in elucidating the pathogenic mechanism of SSPE and developing potential therapeutics.
8. Whitaker, J.W. Effects of Enrichment and Litter Parity on Reproductive Performance and Behavior in BALB/c and 129/Sv Mice / J. Whitaker, S.S. Moy, K.R. Pritchett-Corning, C.A. Fletcher // J Am Assoc Lab Anim Sci. - 2016. – V. 55. – I. 4. – P. 387-399.