CN106420687A - Method for preventing intestinal radiation damage and application of TPA - Google Patents
Method for preventing intestinal radiation damage and application of TPA Download PDFInfo
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- CN106420687A CN106420687A CN201610885517.2A CN201610885517A CN106420687A CN 106420687 A CN106420687 A CN 106420687A CN 201610885517 A CN201610885517 A CN 201610885517A CN 106420687 A CN106420687 A CN 106420687A
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Abstract
The invention discloses a method for preventing intestinal radiation damage, wherein the intestinal stem cell genes Dclk1, Msi1, Lgr5, Bmi1 and Notch1 are up-regulated through TPA, the key signal proteins PKC-betaII and Erk1/2 as well as the protein p90RSK are further up-regulated, and the intestinal epithelial cell proliferation is promoted to prevent intestinal radiation damage.
Description
Technical field
The present invention relates to a kind of protection method of intestinal radiation damage and the purposes of TPA.
Background technology
Today's society, the development and utilization of nuclear energy widely, with the increase of nuclear incident possibility occurrence, is found new
Antiradiation drug, can mitigate to vitals such as bone marrow and the potential detrimental effects of gastrointestinal, it appears particularly necessary with urgent.
In medical field, radiotherapy is to treat one of the most frequently used treatment meanss of malignant tumor, but the untoward reaction that radiotherapy causes is to patient
Huge body and mind injury is brought, the side effect for reducing radiotherapy is still a clinical difficult problem urgently to be resolved hurrily.Gastrointestinal tract and hemopoietic system
To radiation height sensitivity, it is main causes of death after exposure radiation to radiate the gastro-intestinal tract syndrome for causing and bone marrow depression.?
Generally, the cell (intestinal mucosa and medullary cell) of quick division is most susceptible to the injury for radiating, although in radiotherapy skill
Today of art fast development, radioactivity enteropathy is still a key factor for hindering tumor patient to treat further.Data display,
The abdominal part tumor of pelvic cavity patient of 60-80% receives occur intestinal signs of toxicity and bowel dysfunction during radiotherapy.The wind of radioactivity enteropathy
Danger limits the quality of life for treating and reducing cancer patient further of tumor.
The acute damage of intestines that high dose radiation causes, mainly due to the death of intestinal mucosa stem cell.Clinical characters mainly have：
, the incubation period of last from days, there is gastrointestinal symptom afterwards in nausea,vomiting,diarrhea, septicemia, electrolyte imbalance etc., and finally
Dead.Amifostine (free radical scavenger), is to ratify at present, for radiocurable classics medicine, to radiate clinical gastrointestinal is prevented
Toxicity shows certain effect, but side effect is serious and therapeutic time window is narrow.Therefore, development low toxicity, efficient radiation are anti-
Shield agent is necessary.
Therefore, in order to solve many not enough and defects of above-mentioned prior art, it is necessary to which a kind of protection intestinal radiation of research is damaged
The method of wound.
Content of the invention
The present invention is completed in view of at least one the problems referred to above, and it is an object of the present invention to provide protection
The method of intestinal radiation damage, the method for the protection intestinal radiation damage raises small intestinal stem cell gene Dclk1, Msi1 by TPA,
Lgr5, Bmi1 and Notch1, and key signal albumen PKC- β II and Erk1/2, and albumen p90RSK is dialled further up, promote
Proliferation of Rat Intestinal Crypt Cells, protects intestinal radiation damage.
It should be noted that the term " protection " in the present invention is referred to protect and repaired, i.e., dropped by protection and reparation
The radiation damage of low intestinal.
According to a further aspect of the invention, there is provided purposes of the TPA in protection intestinal radiation damage, it is characterised in that according to upper
State method protection intestinal radiation damage.
Compared with prior art, the beneficial effects of the present invention is：
The TPA of the present invention has radiation resistance, prevents radiation damage from causing the mortality of intestinal crypt stem cells, subtracts
Slow body weight reduces and contributes to weight recovery.
Description of the drawings
Fig. 1 is experimental result of the method for the protection intestinal radiation damage according to a kind of preferred embodiment of the present invention on mice
Comparison diagram, the particularly apoptosis rate testing result figure of flow cytometry.
Fig. 2 is another reality of the method for the protection intestinal radiation damage according to a kind of preferred embodiment of the present invention on mice
Results contrast figure is tested, particularly for the impact figure of IEC-6 cell propagation after radiation.
Fig. 3 is another experiment of the method for the protection intestinal radiation damage according to a kind of preferred embodiment of the present invention on mice
Results contrast figure, particularly impact figure of the TPA to mouse survival after radiation and body weight.
Fig. 4 is another experiment of the method for the protection intestinal radiation damage according to a kind of preferred embodiment of the present invention on mice
Results contrast figure, particularly impact figure of the TPA to small intestinal stem cell gene after radiation.
Specific embodiment
Below in conjunction with the accompanying drawings, the preferred forms of the present invention are described, being embodied as here by preferred embodiment
Mode is to explain the present invention, and should not be construed as limiting the invention, in the spirit without departing from the present invention and reality
In the case of matter scope, various changes and modifications can be made, these should all be included within protection scope of the present invention.
Fructus Crotonis L belongs to Euphorbiaceae, originates in the greenery shrub in Southeast Asia.12- oxygen-TPA
(TPA) be the Oleum Tiglii for being extracted from the plant main active.TPA has extensive biologic activity, in animal body
The generation of interior inducible cytokine profiles.TPA has protective effect and to its possible protection machine to acute bowel radiation damage
System is verified further.Experiment in vivo and vitro shows, TPA has radiation resistance, prevents radiation damage from causing intestinal crypt stem cells
Mortality, be conducive to crypts to regenerate, and extend survival time of mice, slow down body weight and reduce and contribute to weight recovery.Additionally,
Observe the different degrees of upper mediation of gene Dclk1, Notch1, Lgr5, Bmi1 and Msi1 related to intestinal crypt stem cells
The activation of PKC, Erk1/2, p90RSK signaling pathway protein, prompting TPA is for small intestinal radiation damage with protection work further
With.
Referring to Fig. 1-3, the invention provides a kind of method of protection intestinal radiation damage.The side of the protection intestinal radiation damage
Method may include：Small intestinal stem cell gene Dclk1, Msi1, Lgr5, Bmi1 and Notch1 are raised by TPA, and is dialled further up closing
Key signals albumen PKC- β II and Erk1/2, and albumen p90RSK, promote Proliferation of Rat Intestinal Crypt Cells, protect intestinal radiation damage.
Preferably, the invention provides a kind of purposes of the also TPA in protection intestinal radiation damage, which passes through preceding method and prevents
Shield intestinal radiation damage.
Embodiment 1
1st, experimental cell and condition of culture
Jejunum in rats epithelial cell line (IEC-6) is attached cell, and its condition of culture is：Using H-DMEM culture medium, contain
10%FBS, 1% is dual anti-, 1U/ml insulin regular, is placed in 37 DEG C, 5%CO2Culture in cell culture incubator, routine changes liquid in three days,
1：3-6 is passed on, and adds 1 milliliter of pancreatin when passing on.Incubator digested 2-3 minute, digest excessively, after the fusion of 80% attached cell
Terminate digestion immediately.All take the logarithm trophophase cell is tested every time.
2nd, laboratory animal and rearing conditions
SPF level BALB/c mouse, female, 6-8 week old, body weight 18-20g, purchased from Beijing dimension tonneau China laboratory animal technology
Company limited, credit number：SCXK (capital) 2012-0001.Degerming rear nursing, drinking water per 5 standard feed ultra-vioket radiation of cage
Freely drink for mice after sterilization.
3rd, radiation and crypts are counted
Irradiation condition：Military Medical Science Institute's Radiation Center60Co gamma-radiation, exposure dose is 10Gy and 7.5Gy.Press
Crypts of small intestine number is counted under Withers HR method mirror.Crypts standards for recycling is:Pit cell number >=10, endochylema are few, basophilic
Property substantially and have Mitotic index, cell in intensive shape.Unrenewable crypts is the dispersion of acellular or cell, increase, endochylema
Acidophilia.The nucleus that pit cell number >=5 are connected, Ki67 is positive.
4th, statistical analysis
Experimental result is represented with mean+SD, and GraphPad Prism6 software is mapped and statistical analysiss.Three
Group data are compared with one factor analysis of variance, p<0.05 represents that difference is statistically significant.
Referring to Fig. 1, IEC-6 cell, radiate latter 48 hours and detect.A：Untreated；B：TPA(1nM)；C：Control；
D：Different group apoptosis comparison diagram (quantitations).Independently repeat experiment for three times.Mean±SEM.**P≤0.01.TPA alleviates spoke
Penetrate the apoptosis of the IEC-6 cell for causing.Radiating first 12 hours and cell is processed with TPA (1nM), is withered with flow cytomery cell
Die situation.At 48 hours, TPA group cell had relatively low apoptosis rate (14.13% ± 0.47%), and cellular control unit apoptosis rate is
(19.83% ± 0.68%), blank control group apoptosis rate is (6.80% ± 0.80%).
Referring to Fig. 2, IEC-6 cell, TPA concentration is 1nM.Three independent repeated trials.Mean±SEM.*P≤0.05；**
P≤0.01.TPA promotes the propagation of IEC-6 cell after radiation.Radiating first 12 hours and cell is processed with TPA (1nM), is examined with CCK-8
Test each group cell proliferative conditions.The ability of TPA group IEC-6 cell propagation was gradually risen in initial 4 days.The IEC-6 of matched group
The ability of cell propagation increases significantly lower than TPA group.
TPA can alleviate the retardance for radiating for cell cycle.Cell is caused to hinder by flow cytomery to radiation
Stagnant in the G1 phase, by taking the 3rd day testing result as an example：It is 45.80% that blank control group cell G1 phase ratio is 41.63%, TPA group,
Matched group is 50.88%, TPA group between blank control group and matched group.4th day consistent with the 3rd day with the 5th day result.
TPA promotes transition of the IEC-6 cell from G1 to the S phase after radiation.
Experiment shows, after TPA promotes radiation, IEC-6 cell turns to the S phase by the G1 phase.First 12 hours are radiated with TPA (1nM) place
Reason cell, uses flow cytomery cell cycle stage.TPA to a certain degree relieves the week of radiation-induced IEC-6 cell
Phase blocks.Radiation causes cell cycle arrest in the G1 phase, and according to flow cytometry analysis TPA group (3-5 days), TPA promotes cell
The S phase is turned to by the G1 phase.
TPA increased the survival of crypts of small intestine.In order to inquire into impact of the TPA to BALB/c mouse Jejunum Crypt after radiation,
The TPA of various dose is administered (treatment group, n=3 in first 3 days continuous tail vein injections of radiation；Matched group, n=3).It is right to observe
According to group mouse survival crypts average out to 9.29 ± 1.8；At TPA optimal dose group (100 μ g/Kg), observe that Surviving crypts are average
For 35.09 ± 2.3.And administration (testing drug repair) in continuous three days after irradiation, then do not find obvious crypts number
Amount increases (data do not show).While 3.5 days after irradiation, when execution mice takes intestinal tube, it can be observed that various dose group is little
The clinical manifestation such as tube wall that intestinal has degree different is thinning, edema and intestinal tube are adhered, and 100 μ g/Kg dosage group intestinal tube edema, glutinous
The performance of the inflammatories such as company is most light.
3.5 days crypts number average out to 35.09 of TPA optimal dose group (100 μ g/Kg), are 3.78 times of matched group.
Referring to Fig. 3, AB：Different group female mice time-to-live comparison diagrams；C：Different group female mice relative body weights
Comparison diagram (the original body mass percentage ratio of measured body weight).Mean±SEM.*P≤0.05；**P≤0.01.TPA optimal dose group
(100 μ g/Kg) median survival interval is 11 days, is 2.2 times of matched group；TPA optimal dose group weight loss is slow compared with matched group, and
Started meaningful rise at the 5th day.
TPA extends the mouse survival time and slows down Mouse Weight decline.Choose the existence week of 7.5Gy dose measurement mice
Phase.Observe control group mice mean survival time for 5.4 days (median survival interval is 5 days), optimal dose group (100 μ g/Kg)
Mice mean survival time is 12.6 days (median survival interval is 11 days).While starting after irradiation to measure body to mice daily
Weight, analytical data result is as follows：From after radiation the 1st day, the body weight of each group mice all declined rapidly, but TPA group compares matched group
Average weight declines more slowly, and the body weight of TPA group mice was from bottom out in the 5th day.Obtaining these results indicate that TPA is obvious
Extend the life cycle of mice and prevent fatal weight loss.
Referring to Fig. 4, IEC-6 cell, TPA concentration is 1nM.Three independent repeated trials.Mean±SEM.*P≤0.05.Real
Test and show, 24 hours after irradiation, detect small intestinal stem cell gene Dclk1, Msi1, Lgr5, Bmi1 and Notch1 is different degrees of
Rise.
TPA increased the expression of small intestinal stem cell gene.Extract RNA within 24 hours after irradiation, tested by PT-PCR, see
Measure generally acknowledged gene Dclk1, Msi1, Lgr5, the Bmi1 and Notch1 of some related to intestinal stem cell significantly to raise (on Dclk1
3.86 times of tune, 2.08 times of Msi1 rise, 1.76 times of Lgr5 rise, Bmi1 raise 2.62 times, 1.94 times of Notch1 rise), and with
Matched group difference is statistically significant.
Experiment shows, after radiation, has done the detection of continuous 4-5 days to signaling pathway protein.TPA group key signal path egg
White PKC- β II and Erk1/2 activation is raised, and pathway protein p90RSK activation was raised, in the 1st day intensity of activation maximum.
Experiment in vivo and vitro confirms, it is thin that 12- oxygen-TPA (TPA) can substantially increase IEC-6
The propagation of born of the same parents, slow down the apoptosis that causes of radiation and cell cycle arrest, and 3.5 days crypts of small intestine survival numbers can be dramatically increased
Measure, extend survival time of mice, delay weight loss.Be detected simultaneously by intestinal crypt stem cells related gene Dclk1, Msi1,
The different degrees of rise of Lgr5, Bmi1 and Notch1, signaling pathway protein PKC, ERK1/2, p90RSK are also in activation accordingly
Adjust.Absolutely prove that TPA has protective effect to acute bowel radiation damage.
Embodiment 2
By case-control study, three groups of radiotherapy group are selected, wherein first group is radiated using amifostine protection intestinal, second
Group is provided without any protection medicine；3rd group uses 12- oxygen-TPA (TPA).Experimental result table
Bright, intestinal mucosa cells mortality rate is respectively 13.9%, 19.9%, 14.1%, the symptom of the acute damage of intestines of patient (Nausea and vomiting,
Diarrhoea) be respectively：Slightly, hence it is evident that, slightly.The side effect of appearance is respectively：Seriously, no, slightly.
In sum, the beneficial effects of the present invention is：
The TPA of the present invention has radiation resistance, prevents radiation damage from causing the mortality of intestinal crypt stem cells, subtracts
Slow body weight reduces and contributes to weight recovery.
The invention is not restricted to above-mentioned specific embodiment.It is understood that without departing from spirit and substance of the present invention model
In the case of enclosing, various changes and modifications can be made, these should all be included within protection scope of the present invention.
Claims (2)
1. a kind of protection intestinal radiation damage method, it is characterised in that by TPA raise small intestinal stem cell gene Dclk1, Msi1,
Lgr5, Bmi1 and Notch1, and key signal albumen PKC- β II and Erk1/2, and albumen p90RSK is dialled further up, promote
Proliferation of Rat Intestinal Crypt Cells, protects intestinal radiation damage.
Purposes of the 2.TPA in protection intestinal radiation damage, it is characterised in that method protection intestinal radiation according to claim 1
Damage.
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Cited By (1)
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| CN115154587A (en) * | 2022-05-31 | 2022-10-11 | 中国人民解放军海军军医大学 | Application of Creld2 protein or gene in prevention and treatment of intestinal injury |
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| CN115154587A (en) * | 2022-05-31 | 2022-10-11 | 中国人民解放军海军军医大学 | Application of Creld2 protein or gene in prevention and treatment of intestinal injury |
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Application publication date: 20170222 |