CN102871770B - Method for establishing hypothyroidism Wistar rat orbit model by adopting radioactive internal radiation - Google Patents
Method for establishing hypothyroidism Wistar rat orbit model by adopting radioactive internal radiation Download PDFInfo
- Publication number
- CN102871770B CN102871770B CN201210388135.0A CN201210388135A CN102871770B CN 102871770 B CN102871770 B CN 102871770B CN 201210388135 A CN201210388135 A CN 201210388135A CN 102871770 B CN102871770 B CN 102871770B
- Authority
- CN
- China
- Prior art keywords
- hypothyroidism
- group
- wistar rat
- internal radiation
- model
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention relates to a method for establishing a hypothyroidism Wistar rat orbit model by adopting radioactive internal radiation. The method comprises the following steps of: (a), performing radioactive 131I internal radiation on a Wistar rat; (b), performing intra-peritoneal injection on the Wistar rat by using human thyrotrophic hormone; and (c), observing distribution of orbit somatostatin receptors. The method has the advantages that the animal model is easy to manufacture, and the method is low in cost and easy to repeat; the success rate is high, 85 percent of rats have hypothyroidism, and the thyroid tissues have the universal characteristic of the hypothyroidism; complete characteristics of thyroid related eye diseases are reflected; the method discovers that the orbit somatostatin receptors are distributed abnormally; and the death rate of animals is low in experiments.
Description
Technical field
The present invention relates to a kind of construction method of animal model, specifically, is the method adopting radioactivity internal radiation to set up hypothyroidism Wistar rat eye socket model, for observing the distribution of eye socket receptor and expressing.
Background technology
Carry out clinically
131i treats in GD hyperthyroidism process as there is hypothyroidism, very likely there is the danger that TAO worsens, some patients may bring out the generation of Graves oculopathy (GO), its pathogenesis is still not fully aware of, prevention and therapy is all more thorny, there is no proper animal model at present in order to observe the pathological change of this kind of eye socket tissue.
Find for a long time always
131after I treats GD, hypothyroidism brings out the reason of GO, and understands its pathogenesis by animal model observation eye socket histological change, and existing multiple method has made the animal model of hyperthyroidism or hypothyroidism and associated ophthalmopathy thereof at present.
In the selection of one, first laboratory animal, mainly rat, because of the thyroid function of rat and hypothalamic-pituitary-thyroid axis and mankind's basic simlarity, and it is cheap, distribution is wide, breeding is fast, volume is little, be easy to feeding and management.
Two, conventional hypothyroidism model:
1, the method for animal model such as making such as low iodine hypothyroidism model: Fang Hui etc. is: the feedstuff that Wistar Mus eats the grain preparation of severe iodine deficient area adds deionized water, can become mould after March.
2, chemical induction hypothyroidism model: successfully confirm that following chemicals mass-energy brings out hypothyroidism: propylthiouracil (PTU)/iopanoic acid (IOP), PTU, methylthiouracil (MTU), thiamazole (MZ), thyroxin (TH),
131i, sodium perchlorate (NaClO
4).
Rebagliati etc. get the male Mus of 240 ~ 280g Wistar, lumbar injection
131i January, modeling success.
131i method passes through
131i physical action, its mechanism is mainly launches β irridiation injury thyroid cell, thyroxine (TH) is secreted and reduces.
3, congenital hypothyroidism model: both can use iodine-deficient forage, also can bring out with chemical inducer, be pregnant female Mus unlike selected animal.
4, transgenic hypothyroidism model.
5, thyroidectomy hypothyroidism model: thyroidectomy hypothyroidism model is classical hypothyroidism model, excises most thyroid and must cause hypothyroidism.
Above hypothyroidism model is used for hyperthyroidism warp
131i is still undesirable, does not simulate the naturalness of disease progression well.Simultaneously because the hypothyroidism cause of disease is complicated, current hypothyroidism animal model does not also include the hypothyroidism of all causes of disease, is mostly limited to the research of hypothyroidism and TH opposing syndrome, resists syndrome then study very few for secondary hypothyroidism and TSH.
Three, method contrast is summed up.
Low iodine hypothyroidism model (comprising low iodine Congenital Hypothyroid model) exist consuming time, the shortcoming such as to expend.
Chemical induction hypothyroidism model (comprising chemical induction hypothyroidism model) exist consuming time, cost, can not the better shortcoming such as simulating nature course of disease.
There is operating process complexity, technical strong, the shortcoming such as success rate is low in transgenic hypothyroidism model.
Although excision hypothyroidism model is simple to operation, modeling success rate Gao Keda 100 %, is not the natural hypothyroidism after Treatment of Hyperthyroidism.
The oculopathy that above model all causes hypothyroidism with actual hyperthyroidism through internal radiation compares, and its inducement and evolution have larger difference.In addition, between each model, the successful standard of modeling is also different.Should be different according to research purpose, manufacture different hypothyroidism animal models.Possess simultaneously consuming timely to expend less, simple to operate, success rate is high, the condition of the naturalness of simulation disease progression is only desirable hypothyroidism animal model.Directly adopt the method for internal radiation hyperthyroidism animal to cause the eye in orbit,phantom of hypothyroidism at present, not yet find bibliographical information both at home and abroad.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of method adopting radioactivity internal radiation to set up hypothyroidism Wistar rat eye socket model is provided.
For achieving the above object, the technical scheme that the present invention takes is: adopt radioactivity internal radiation to set up the method for hypothyroidism Wistar rat eye socket model, described method comprises the following steps: a, pass through radioactivity
131i internal radiation Wistar rat; B, end user's TSH-like lumbar injection Wistar rat.
Radioactivity is passed through in described step a
131the method of I internal radiation Wistar rat is lumbar injection
131i sodium iodide.
Described
131the lumbar injection dosage of I sodium iodide is that 300 μ ci or 500 μ ci/ are only every.
The time of described people's TSH-like lumbar injection is the 6th week.
Described method comprises the following steps: a, lumbar injection
131i sodium iodide injection, injected dose is that 300 μ ci or 500 μ ci/ are only every; B, the 6th week, people's TSH-like with 0.9% normal saline dilution to 83.2 μ g/mL, rats by intraperitoneal injection, injected dose is that 100 μ L/ are every only; C, for observing the distribution of eye socket somatostatin receptor.
The invention has the advantages that:
1, this animal model makes simple, and expense is low, easily repeats;
2, success rate is high, and 85% hypothyroidism occurs, parathyroid tissue and have the universals of hypothyroidism;
3, wherein 100% feature having a thyroid-associated ophthalmopathy;
4, eye socket somatostatin receptor abnormal distribution is found;
5, in test, mortality of animals is low.
Accompanying drawing explanation
Fig. 1. eye socket organizes extraocular muscles pathological change, D. normal group × 100; B. middle dosage group × 100, arrow indication, mucinous degeneration, edema, fatization; C. high dose group × 100, arrow indication, muscle fiber is transparent, degeneration fracture.
Fig. 2. eye socket extraocular muscles somatostatin receptor (SSTR2,5) SABC (arrow indication is positive cell),
normal group SSTR2 × 400;
high dose group SSTR2 × 400;
normal group SSTR5 × 400;
high dose group SSTR5 × 400.
Detailed description of the invention
Below in conjunction with accompanying drawing, detailed description of the invention provided by the invention is elaborated.
Embodiment
The object of the invention is to be expert at radioiodine-131(by simulation hyperthyroidism (hyperthyroidism)
131i) method of internal radiation, sets up a kind of simple and easy effective Hypothyroidism (hypothyroidism) eye socket animal model, and pathological manifestations, similar in appearance to thyroid-associated ophthalmopathy (TAO), needs for observation and discussion TAO pathogenesis.The present invention passes through
131i lumbar injection Wistar rat, then adopts the higher people's TSH-like (hTSH) of purity to stimulate Wistar rat, Wistar rat generation hypothyroidism, observes animal thyroid, eye socket tissue, judgment models whether successfully.
one, materials and methods
1 material
1.1 animal 20 Wister rats (male and female half and half, average weight 220g), are provided by Fudan University's institute of radio-medicine's animal experimental center and are raised.
1.2 main agents
131i-sodium iodide injection (Xinke Medical Co., Ltd., Shanghai); FT
3, FT
4test kit (ROCHE diagnostic products Shanghai company limited); Wister rat thyrotropin (TSH) elisa kit and mouse-anti people somatostatin receptor polyclonal antibody (abbreviation somatostatin antibody) 2 and 5 purchased from American ADL company; SP group test kit and DAB colour reagent box are purchased from Shanghai Long Island company.
1.3 instrument COBAS E601 Electrochemiluminescince immunoassay instrument (ROCHE diagnostic products Shanghai company limited), Multskan MS microplate reader (Germany).
2 methods
The method of 20 Wister rat completely randomized designs is divided into normal group (D group) 6, experimental group 14 with disposal by 2.1 experimental animal groupings, and rat tail vein gets blood 0.5mL, measures FT
3.Experimental group lumbar injection
131experiment component is three groups according to various dose (only every use 100 μ ci, 300 μ ci, 500 μ ci): low dose group (A group) 4, middle dosage group (B group) and each 5 of high dose group (C group) by I-sodium iodide injection.6th week, hTSH to 83.2 μ g/mL, got 100 μ L(containing 0.0832 μ g with 0.9% normal saline dilution) rats by intraperitoneal injection.In injection
131within after I the 8th week, put to death all rats, heart extracting blood 2mL, it is to be measured that centrifuging and taking supernatant places-40 DEG C of refrigerators.Carefully peel off eye socket tissue with shears and tweezers, put into 10% neutral formalin immediately liquid-solid fixed.
2.2 serum FT
3, FT
4, TSH measure use ElectrochemiluminescDetermination Determination FT
3, FT
4, ELISA measures rat blood serum TSH.
Fixing is organized ethanol serial dehydration, specimens paraffin embedding slices by 2.3 eye socket pathological study, and haematoxylin & eosin (HE) dyes, and observes under an optical microscope.
2.4 immunohistochemical method tissue dewaxings are to water, and after antigen hot repair is multiple, tissue slice surface drips somatostatin antibody 2 and 5(working solution concentration is 1:100), be placed in wet box, 4 DEG C of fridge overnight.Replace primary antibodie as negative control with PBS.After hatch through SP working solution, PBS wash, DAB develop the color, haematoxylin is redyed.Antibody staining positive reaction product is positioned on Cytoplasm and cell membrane, in brown granular shape, to occur evenly completely paintedly being decided to be the positive.
1.3 statistical method
With statistical analysis software: application SPSS13.0 statistical package, all experimental datas all take mean ± standard deviation (
± S) represent.Different group compares employing variance analysis,
p<0.05 is that difference has statistical significance.
two, result
Wistar rat blood serum FT
3, FT
4and compare (table 1) between rTSH horizontal group.
Table 1 Wistar rat blood serum FT
3, FT
4and compare between rTSH horizontal group
Note: compare with D group ▲
p<0.05; Δ is compared with A group
p<0.05.
Before experiment, Wister rat blood serum FT
3no significant difference between each group of level (
p>0.05).Experimental group is injected
131i is after 8 weeks, serum FT
4level obviously declines, and compares with normal group, difference all have statistical significance (
p<0.05), wherein B group and C group be starkly lower than A group (
p<0.05), difference all have statistical significance (
p<0.05); Serum TSH level, B group, C group a little more than normal group, A group a little less than normal group, the equal not statistically significant of difference (
p>0.05).
(Fig. 1) is observed in 2.1 HE dyeing
Low dose group: mucinous degeneration, edema appear in about 25% eye socket tissue, and without obvious fibrous tissue and adipose tissure hyperplasia, muscle fiber ruptures without degeneration.Middle dosage group: obvious mucinous degeneration, edema appear in about 50% rat eye socket tissue, local fiber tissue and adipose tissure hyperplasia, muscle fiber hyaline degeneration ruptures; High dose group: obvious mucinous degeneration, edema appear in about 70% rat eye socket tissue, local fiber tissue and adipose tissure hyperplasia, muscle fiber hyaline degeneration ruptures, and cavity is formed.In addition, experimental group all has mastocyte to be in various degree main immune cell infiltrate.Normal group: the eye socket tissue of mice is all without morphological change.
2.2 ImmunohistochemistryResults Results judge (Fig. 2)
SSTR positive staining is brown yellow granule, is positioned cell membrane and cytoplasm.Often open section Stochastic choice 10 high power fields, ratio according to positive cell judges, 20% ~ 50%SSTR positive is decided to be the weak positive (+), be greater than 50% and be decided to be the positive (++), be greater than 75% and be decided to be strong positive (+++), be less than the cell color of 20% or cell is consistent with background is not paintedly decided to be feminine gender (-).
D group 6: SSTR2 is 3/6 (-), 3/6 (+), and SSTR5 is 2/6 (-), 3/6 (+), 1/6 (++); A group 3: SSTR2 is 2/3 (+), 1/3 (+++), SSTR5 is 2/3 (+), 1/3 (+++); B group 8: SSTR2 is 5/8 (+), 3/8 (+++); SSTR5 is 3/8 (+), 5/8 (+++); C group 6: SSTR2 is 5/6 (+), 1/6 (+++); SSTR5 be 6/6 (+++).A group and B group are merged into AB group, passes through
χ 2inspection or correction
χ 2inspection, the expression of SSTR 5 C group, D group and AB group three group difference have statistical significance (
p< 0. 0005), and the differential expression not statistically significant of SSTR 2 (
p> 0. 05).
three, discuss
SSTRs is prevalent in each histiocyte of human body, finds that SSTR has 5 hypotypes at present.Different cell surface is organized all to find that there is SSTRs at TAO patient's eye socket, lymphocytic cell surface is containing all SSTR hypotypes, but SSTR1-3 expresses relatively strong, in orbital fat cell, express SSTR1-3 and SSTR5 in fibrous tissue, extraocular muscles cell only has the expression of SSTR1-3.Existing multinomial research finds, in TAO active stage, it is relevant to its mobility that the lymphocyte of Qiu Hou tissue infiltration and the fibroblast of activation express SSTR quantity.The cytological effect of SSTR mediation is more, and be mainly manifested in the secretion of T suppression cell and cell differentiation and cell death inducing, SSTR also can suppress the lymphocyte release cells factor, immunity moderation cell function.
The video picture of SSTA eye socket combines based on the lymphocyte of SSTA and the Qiu Hou tissue infiltration by radioisotope labeling and the fibroblast SSTRs of activation, effectively reflects the activeness of eye socket tissue, and guide the treatment to patient.The mechanism that SSTA is used for the treatment of Graves oculopathy still imperfectly understands, possible mechanism has following 3 possibilities: one is the activity by suppressing insulin like growth factor-1 (IGF-I), thus reduce IGF-I to fibroblastic stimulation, inhibit the synthesis of glycosaminoglycan (GAG); Two is the materials by suppressing the release cells factors such as T lymphocyte or promoting immunity inflammatory reaction; Three is be combined by the SSTR directly on target cell and cause a series of change, finally reaches the object for the treatment of.
TAO is not only concurrent in GD hyperthyroidism,
131also can exist when there is hypothyroidism after I treatment, the deterioration of its oculopathy strengthens relevant with the sensitivity of tissue T SH receptor after ball.Whether possessed the pathologic basis of TAO generation to separate rat when hypothyroidism occurs, the present invention adopts the methods such as Rebagliati and is improved, and observes the distribution situation of rat extraocular muscles pathological change and SSTR2 and 5 after hypothyroidism occurs.Result shows, obvious mucinous degeneration, edema is there is in rat eye socket tissue, local fiber tissue and adipose tissure hyperplasia, the phenomenons such as muscle fiber hyaline degeneration fracture, and increase the weight of along with the increase of radioactive dosage, and SSTR5 is obviously greater than SSTR2 in the intensity of extraocular muscles expression SSTR, and increase progressively along with the increase of radioactive dosage.
The animal model that the present invention sets up has the pathological characters of hypothyroidism.Through doses
131i internal radiation causes normal rat generation hypothyroidism, and the pathological change of its eye socket tissue is extremely similar to mankind TAO, infer by
131hypothyroidism patient caused by I treatment, the pathological change of TAO also may individualism.Result of study of the present invention thinks that the expression of SSTRs is closely related with the degeneration of eye socket tissue, edema and hypertrophy generally, can provide foundation for the treatment of SSTA or video picture, Estimating curative effect etc.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (1)
1. adopt radioactivity internal radiation to set up the method for hypothyroidism Wistar rat eye socket model, it is characterized in that, described method comprises the following steps: a, lumbar injection
131i sodium iodide injection, injected dose is that 300 μ ci or 500 μ ci/ are only every; B, the 6th week, people's TSH-like is with 0.9% normal saline dilution to 83.2 μ g/mL, and rats by intraperitoneal injection, injected dose is that 100 μ L/ are every only.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210388135.0A CN102871770B (en) | 2012-10-15 | 2012-10-15 | Method for establishing hypothyroidism Wistar rat orbit model by adopting radioactive internal radiation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210388135.0A CN102871770B (en) | 2012-10-15 | 2012-10-15 | Method for establishing hypothyroidism Wistar rat orbit model by adopting radioactive internal radiation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102871770A CN102871770A (en) | 2013-01-16 |
| CN102871770B true CN102871770B (en) | 2015-06-03 |
Family
ID=47473456
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210388135.0A Active CN102871770B (en) | 2012-10-15 | 2012-10-15 | Method for establishing hypothyroidism Wistar rat orbit model by adopting radioactive internal radiation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102871770B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0212522A2 (en) * | 1985-08-12 | 1987-03-04 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Method for the measurement of TSH |
| CN1943723A (en) * | 2006-10-23 | 2007-04-11 | 大庆龙南医院 | A Chinese traditional medicinal embolus for treatment of hyperthyroidism and its preparation method |
-
2012
- 2012-10-15 CN CN201210388135.0A patent/CN102871770B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0212522A2 (en) * | 1985-08-12 | 1987-03-04 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Method for the measurement of TSH |
| CN1943723A (en) * | 2006-10-23 | 2007-04-11 | 大庆龙南医院 | A Chinese traditional medicinal embolus for treatment of hyperthyroidism and its preparation method |
Non-Patent Citations (2)
| Title |
|---|
| 《131I治疗后Graves眼病血清TNF-α、sTNFRⅡ和sICAM-1的相关性研究》;褚俏梅等;《临床眼科杂志》;20111231;第19卷(第4期);316-319 * |
| 《因宁片对实验性甲亢阴虚大鼠的药效学研究》;张薇等;《中药材》;20070331;第30卷(第3期);326-328 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102871770A (en) | 2013-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Eusemann et al. | Influence of a sustained release deslorelin acetate implant on reproductive physiology and associated traits in laying hens | |
| Marley et al. | Effect of forage legumes and anthelmintic treatment on the performance, nutritional status and nematode parasites of grazing lambs | |
| CN111149767B (en) | Construction method and application of humanized skin type lupus erythematosus mouse model | |
| CN106857406A (en) | A kind of method for building up by diet induced SD rat diabetes animal models | |
| Campos et al. | Effect of obesity on rat reproduction and on the development of their adult offspring | |
| Ritter | XL the effect of hypothyroidism on the larynx of the rat: an explanation for hoarseness associated with hypothyroidism in the human | |
| CN103830721B (en) | A kind of derivant and preparation and application thereof of setting up rheumatoid arthritis animal model | |
| CN102871770B (en) | Method for establishing hypothyroidism Wistar rat orbit model by adopting radioactive internal radiation | |
| Brito et al. | Induction of Zenk protein expression within the nucleus taeniae of the amygdala of pigeons following tone and shock stimulation | |
| US9186421B2 (en) | Establishment of rhesus monkey model of autoimmunity type 1 diabetes | |
| Day | Ocular manifestations of thyroid disease: current concepts | |
| CN102517333A (en) | Spontaneous HHcy-related atherosclerosis transgenic mice animal model and preparation method thereof | |
| Bompadre et al. | Trichostrongylus colubriformis infection negatively impacted phosphorus metabolism of lambs | |
| CN106443024B (en) | LGR4 genes/proteins are used to screen the drug for the treatment of hypercholesterolemia | |
| Boussinesq et al. | Studies on the macrofilarial population of Onchocerca volvulus in hyper-endemic villages of the Central province of Cameroon | |
| Tunbridge et al. | Primary hypothyroidism presenting as pituitary failure. | |
| Bartlett et al. | Diagnosis and management of diabetes mellitus in a Bali mynah (Leucopsar rothschildi) | |
| CN103173404A (en) | Establishing method of hypertensive disorder complicating pregnancy mouse model | |
| Wang et al. | Clozapine Induces Myocardial Inflammation Response Through Lactation Modification---Based on Novel Tree Shrew Models of Schizophrenia | |
| Sahinduran et al. | Necrolytic migratory erythema in a cat with glucagonoma syndrome | |
| Pantoja et al. | Urinary parameters of Trichechus inunguis (Mammalia, Sirenia): reference values for the Amazonian Manatee | |
| Ewuola et al. | RELATIONSHIP AMONG EXTERNAL MORPHOMETRIC TRAITS AND SEMEN QUALITY IN BREEDER COCKS AND EGG CHARACTERISTICS IN LAYING HENS | |
| Yuen | An assessment of reproductive development of the male indo-Pacific bottlenose dolphin, Tursiops aduncus, in captivity | |
| Stewart et al. | Administration of low and high doses of heparin causes changes in plasma non-esterified fatty acid concentration in merino and terminal sired lambs | |
| Ferreira | Analysis of neutrophil recruitment during tissue regeneration in zebrafish models of human disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |