CN101732332B - Application of 20(S)-ginsenoside Rg3 in preparation of medicines for treating non-small cell lung cancer - Google Patents
Application of 20(S)-ginsenoside Rg3 in preparation of medicines for treating non-small cell lung cancer Download PDFInfo
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Abstract
The invention relates to medicine application of 20(S)-ginsenoside Rg3, in particular to application of 20(S)-ginsenoside Rg3 in preparation of medicines for treating a non-small cell lung cancer. In the invention, selecting a human non-small cell lung cancer cell strain (A549 lung adenocarcinoma, H460 large cell lung cancer and LTEP-78 lung squamous carcinoma) to be inoculated under the skin of a naked mouse, and observing the influence of the SPG-Rg3 on the non-small cell lung cancer; and carrying out CD34 immunohistochemistry staining and TUNEL immunofluorescence staining on tumor tissues, and observing the influence of the SPG-Rg3 on tumor angiogenesis and apoptosis. The inclusion proves that the 20(S)-ginsenoside Rg3 can remarkably inhibit the growth of the non-small cell lung cancer, has the action mechanism related with the promotion of the tumor apoptosis and the inhibition of the tumor angiogenesis and unobvious cooperation action when being combined with cyclophosphamide for application, and can be used for the clinical chemotherapy of the non-small cell lung cancer.
Description
Technical field
The invention belongs to the drug research field, specifically, relate to 20 (S)-ginsenoside Rg3s as the application in the medicine of preparation treatment nonsmall-cell lung cancer.
Background technology
Pulmonary carcinoma is clinical common cancer, usually be divided into small cell lung cancer and nonsmall-cell lung cancer two big classes, its incidence and mortality is all first of the row malignant tumor, nonsmall-cell lung cancer (non-small cell lung cancer wherein, NSCLC) account for the overwhelming majority, the most general, account for more than 80% of lung cancer morbidity sum.Nonsmall-cell lung cancer rises year by year at global sickness rate, and human health in serious threat.The method of treatment NSCLC mainly contains following several at present:
(1) NSCLC surgical intervention
The optimal method of treatment NSCLC is still surgical operation therapy at present.The purpose of operative treatment is excision pulmonary carcinoma focus and regional lymph nodes, and keeps healthy lung tissue as far as possible.At present the argon helium knife cryoablation is as Minimally Invasive Surgery, is applicable to that focus is less, body constitution is relatively poor, is difficult to the patients with lung cancer that tolerates open chest surgery or disagree with capable open chest surgery.
But surgical operation is the major measure of early stage NSCLC treatment, and the surgery alone long term survival still can not be satisfactory, and 5 years survival rates of IA phase patient have only 70%, then reduce to 17% to the Ш A phase, analyze the reason of operative failure, majority is a metastasis, and prompting needs whole body therapeutic.
(2) chemicotherapy
At present, radiotherapy NSCLC is the most common except that operative treatment, effective topical therapeutic means.The restriction ratio operation of aspects such as it is dissected, stadium, muscle power is little, is accepted by the patient easily.
Chemotherapy is widely used in the treatment of pulmonary carcinoma, can be applied to the advanced lung cancer case separately, plays the palliative treatment effect, and mostly is as therapeutic alliances such as the part of Comprehensive Treatment and radiotherapy, operations.Chemotherapy can prevent that cancerous protuberance from shifting, recurrence, improves long-term survival rate.Based on the chemotherapy of cisplatin (DDP) can be that the patient brings existence to benefit behind the complete excision, especially ∏ phase and Ш phase patient; The gerontal patient also can obtain the improvement of life cycle from adjuvant chemotherapy.But generally do not advocate adjuvant chemotherapy for IA phase patient; IB phase patient, the status of postoperative adjuvant chemotherapy remains at present in dispute; Usually adopt adjuvant chemotherapy clinically for the above IB of 4cm phase patient.Standard care measure for local advanced NSCLC (Ш A-Ш B) still is synchronous chemicotherapy.Chemotherapy can reduce neoplasm staging and improve the excision rate before the art, combined chemotherapy is to treat NSCLC a kind of means commonly used at present, the compound mode of medicine also has multiple, is the preferred option for the treatment of at present with platinum class associating new drug, and these new drugs comprise gemcitabine, vinorelbine, paclitaxel etc.The local advanced NSCLC of discoveries such as Hanna is accepted EP scheme chemotherapy combined synchronization radiotherapy (54.9Gy), Docetaxel consolidates chemotherapy group PFS and MST was respectively 12.3 months and 21.6 months, all 12.9 months and 24.2 months than simple synchronization combination radiotherapy group descend to some extent, and observe 5.5% toxicity associated death, admission rate also obviously increases, consolidate chemotherapy after the radiotherapy of prompting synchronization and only increase toxic reaction, do not improve overall existence.
(3) molecular targeted treatment
Molecular targeted treatment is meant and utilizes the difference on the molecular cytobiology between tumor cell and the normal cell, adopt the sealing receptor, suppress methods such as angiogenesis, disabling signal pathway, act on the specific target spot of tumor cell, suppress the growth of tumor cell specifically, impel apoptosis of tumor cells.Molecular targeted treatment has higher selectivity than traditional chemotherapy, so toxic and side effects is little, is the new trend of oncotherapy from now on, awaits further research and development.
(4) immunization therapy
Immunization therapy mainly is divided into active immunity treatment and passive immunotherapy.
(5) interventional therapy
It is increasingly mature in recent years to get involved bronchus, the intracavitary therapy method is progressively perfect, through fibre bronchus mirror's row laser, microwave, freezing, built-in support, after radiotherapy and high frequency electric knife, argon plasma, balloon expandable etc. in the dress, treat the central airway stenosis or occlusion that tumor causes.To being secondary to the superior vena cava syndrome patient of pulmonary carcinoma, the built-in support of blood vessel is a method preferably.Can improve the tumor by local drug level through the arteriae bronchiales perfusion chemotherapy, the Detoxication that can improve cancer therapy drug simultaneously.
(6) gene therapy
Gene therapy is meant people's normal gene or medicative gene is imported people's somatic target cell by certain way, with correction genetic flaw or performance therapeutical effect, thereby reaches the biomedical technology for the treatment of the disease purpose.
But above-mentioned NSCLC Therapeutic Method remains in some shortcomings, and is ripe not enough as technology, and its curative effect is difficult to confirm; The process of treatment is suffered untold misery, the patient is difficult to bear; Also there are some bigger toxic and side effects in some medicine in treatment, increase patient's misery etc.
The treatment characteristics of Chinese medicine see for integral body and dialectical opinion is controlled, emphasize that topical therapeutic combines with whole body therapeutic, setting upright treatment combines with anticancer therapy, one of means of Comprehensive Treatment had both been can be used as, can be used as a kind of multifactorly in conjunction with the prescription therapeutic on the basis again, have many target spots and take into account advantage with individualized treatment comprehensively, is a kind of Comprehensive Treatment, and the patient need not to bear huge advantages such as misery during with respect to the other treatment scheme, having safely, having no side effect, treat.
Radix Ginseng is the famous and precious Chinese herbal medicine of China, be described as the medical herbs treasure, kind of ginsenoside monomer composition surplus from Radix Ginseng, having isolated 40 at present, wherein the ginsenoside Rg3 belongs to protopanoxadiol type saponin, there are two kinds of homotype isomers, i.e. 20 (S)-ginsenoside Rg3s (SPG-Rg3) and 20 (R)-ginsenoside Rg3s (RPG-Rg3).Studies show that, 20 (S)-ginsenoside Rg3s have anti metastasis effect referring to hot grain husk, Ni Jingsong, Wang Xinrui, the effect [J] that waits the anti-B16 melanoma of .20 (S)-ginsenoside Rg3 to shift. Jilin University's journal; Medicine, 2004,30 (4): 540-543.}, simultaneously 20 (S)-ginsenoside Rg3s also have neoplasm growth effect referring to hot grain husk, Ni Jingsong, Jiang Xin, Wang Xinrui waits .20 (S)-ginsenoside Rg3 to suppress the effect [J] of tumor growth. Jilin University's journal; Medicine, 2006,32 (1): 61-63,81.}.
The Chinese patent of ZL98103433.0 discloses the semisynthesis and the medical usage thereof of 20 (S)-panaxoside Rgs 3.This patent disclosure by from ginsenoside's glycol group as semi-synthetic raw material, adopt chemical method, through semi-synthetic and purify and obtain 20 (S)-panaxoside Rgs, 3 pure product, and disclose this material and be particularly suitable for treating pulmonary carcinoma, melanoma, S in preparation
180Application in sarcoma, colon cancer and the antiviral drug.
Application number is that 200310114432.7 Chinese patent application discloses a kind of to be effective ingredient with 20 (S)-ginsenoside-Rg3, to be used to strengthen the cancer therapy drug curative effect, reduces the medicine of the leucocytes reduction that toxicity of anticancer agents, prevention and treatment chemicotherapy cause; Also provide 20 (S)-ginsenoside-Rg3 to be used to strengthen the cancer therapy drug curative effect simultaneously, the purposes of the leucocytes reduction that reduction toxicity of anticancer agents, prevention and treatment chemicotherapy cause.
Though existingly in the prior art suppress neoplasm metastasis and suppress the relevant report of tumor growth, yet there are no report for the research of nonsmall-cell lung cancer about 20 (S)-ginsenoside-Rg3.Do not find that up to this point a kind of medicine can treat nonsmall-cell lung cancer effectively yet.Whether inhibited the inventor find that 20 (S)-ginsenoside-Rg3 have the effect of obvious inhibition people nonsmall-cell lung cancer growth, thereby have finished the present invention in carrying out 20 (S)-ginsenoside-Rg3 research to the growth of people's nonsmall-cell lung cancer.
Summary of the invention
The object of the present invention is to provide 20 (S)-ginsenoside Rg3s as the application in the medicine of preparation treatment nonsmall-cell lung cancer.
Because 20 (S)-ginsenoside Rg3s' extraction process and other some medicinal usages are open in the prior art, and major technique content of the present invention is the effects of checking 20 (S)-ginsenoside Rg3s on indication of the present invention, therefore, 20 (S)-ginsenoside Rg3s' of the present invention extraction process can be referring to the disclosed content of ZL98103433.0.The present invention chooses people's non-small cell lung carcinoma strain (NCI-H460 National People's Congress cell lung cancer, A549 human lung adenocarcinoma and LTEP-78 people's lung squamous cancer) and is inoculated in BALB/C-nu pure lines nude mice respectively and makes lotus tumor model, animal be will inoculate and blank group, positive controls (cyclophosphamide will be divided into immediately, 40mg/kg/ time, successive administration 5 days), SPG-Rg3 treatment group (3.0mg/kg, 1.0mg/kg, 0.3mg/kg, successive administration, before off-test) and the drug combination group [cyclophosphamide+SPG-Rg3 (20mg/kg+0.5mg/kg)/time, successive administration 5 times].Experiment finishes the back and measures gross tumor volume, weight, calculates tumour inhibiting rate, has observed the inhibitory action of 20 (S)-ginsenoside Rg3s to people's nonsmall-cell lung cancer, concrete visible following embodiment; By embodiment, can further understand the present invention.
By animal experiment, show that 20 (S)-ginsenoside Rg3s can obviously suppress the growth of nonsmall-cell lung cancer, for the medicine that 20 (S)-ginsenoside Rg3s is developed to a kind of people's of treatment nonsmall-cell lung cancer provides foundation fully.
Simultaneously, clinical trial shows that the nonsmall-cell lung cancer patient can obviously suppress the growth of nonsmall-cell lung cancer after adopting 20 (S)-ginsenoside Rg3s treatment, improves patient's quality of life, prolongs patient's life cycle etc.
Description of drawings
Fig. 1 is the influence of SPG-Rg3 to A549 adenocarcinoma of lung growth of xenografted
Fig. 2 is the influence of SPG-Rg3 to H460 maxicell pulmonary carcinoma transplanted tumor volume
Fig. 3 is the influence of SPG-Rg3 to LTEP-78 lung squamous cancer growth of xenografted
Fig. 4 is the inhibitory action of SPG-Rg3 to the growth of A549 adenocarcinoma of lung
Fig. 5 is the inhibitory action of SPG-Rg3 to the growth of NCI-H460 lung large cell carcinoma
Fig. 6 is the inhibitory action of SPG-Rg3 to the squamous cell lung carcinoma growth
Fig. 7 is NCI-H460 lung large cell carcinoma CD34 immunohistochemical staining (400 *) blank group
Fig. 8 is a NCI-H460 lung large cell carcinoma SPG-Rg3 treatment group CD34 immunohistochemical staining (400 *)
Fig. 9 is an A549 adenocarcinoma of lung blank group CD34 immunohistochemical staining (400 *)
Figure 10 is a coloured picture 7A549 adenocarcinoma of lung SPG-Rg3 treatment group CD34 immunohistochemical staining (400 *)
Figure 11-A is A549 blank group TUNEL immunofluorescence dyeing result
Figure 11-B is A549 SPG-Rg3 treatment group TUNEL immunofluorescence dyeing result
Figure 11-C is H460 blank group TUNEL immunofluorescence dyeing result
Figure 11-D is H460 SPG-Rg3 treatment group immunofluorescence dyeing result
The specific embodiment
Below be the specific embodiment of the present invention, described embodiment is in order to further describe the present invention, rather than restriction the present invention.
What present embodiment related to is the research that 20 (S)-ginsenoside Rg3s suppress nonsmall-cell lung cancer growth effect.One, experiment material and method
(1) main material
1. laboratory animal: BALB/C-nu pure lines nude mice is female, 4-6 age in week, body weight 18～22 grams.Purchase respectively in Institute of Experimental Animals, Chinese Academy of Medical Sciences and Beijing Vital River Experimental Animals Technology Co., Ltd..The quality certification number: SCXK (capital) 205-0013 and 2007-0001.Experiment and raising are all carried out in the ultra-clean laminar-flow rack under the SPF condition, and the water of sterilization treatment and feed supply animal are freely taken in, and all operations all carries out in aseptic superclean bench.
2. select the tumor strain for use: NCI-H460 National People's Congress sclc cell line, purchase in Chinese Academy of Sciences's Shanghai cell bank A549 human lung adenocarcinoma and set up by Beijing lung tumors institute by the Peking University's basis present LTEP-78 people of immune teaching and research room of institute Lung Squamous Carcinoma Cells strain
3. experiment reagent: (1) 20 (S)-ginsenoside Rg3, colourless solution, concentration is 3gL-1.Provide lot number 980301 by the Bethune of Jilin University medical college new drug research chamber.Be desired concn with aseptic sodium chloride injection (0.9%) dilution when using.
(2) positive control drug---cyclophosphamide, the 200mg/ bottle, the accurate word (1995) of medicine is defended No. 012034 in Shanghai, Hualian Pharmaceutical Co., Ltd., Shanghai.
(3) negative control medicine---aseptic sodium chloride injection (0.9%), the accurate word H22022845 of traditional Chinese medicines, Cologne, Jilin Cornell pharmaceutical Co. Ltd.0.2ml/ it is inferior.
(4) cell culture fluid---H-DMEM culture fluid (purchasing company) in GIBCO, standard hyclone (Hao ocean, Tianjin).
4. key instrument: SW-CJ-1F superclean bench (SuZhou Antai Air Tech Co., Ltd.); CO2 incubator (Sanyo).
(2) test method
1. animal inoculation: get selected human oncocyte's strain cell of In vitro culture exponential phase, it is subcutaneous to be inoculated in the right axil of BALB/C-nu pure lines nude mice, every mouse inoculation 2 * 10
7Individual cell.Behind the inoculated tumour cell, (the about 0.5cm of diameter, the not obvious person of tumor tuberosity eliminates) is divided into 6 groups at random with mice, 8 every group when treating to touch clear and definite tumor tuberosity.Beginning lumbar injection successive administration, drug withdrawal was put to death animal after 24 hours.Cyclophosphamide is administration every other day, gives 5 times altogether.
1.2 squamous cell lung carcinoma:
2. laboratory animal grouping: be divided into 6 groups behind the nude inoculation oncocyte at random:
1. blank group: intraperitoneal injection of saline 0.2ml/ time.
2. positive controls: intraperitoneal injection of cyclophosphamide 40mg/kg/ time, continuous 5 days.
3. SPG-Rg3 low dose group: 0.3mg/kg, volume: 0.1ml/kg.
4. dosage group: 1.0mg/kg among the SPG-Rg3, volume: 0.1ml/kg.
5. SPG-Rg3 high dose group: 3.0mg/kg, volume: 0.1ml/kg.
6. drug combination group: cyclophosphamide+SPG-Rg3 (20mg/kg+0.5mg/kg).
3. route of administration: lumbar injection, once a day, successive administration finishes the previous day until experiment.
4. observation index:
4.1 periodic measurement mice body weight also writes down the ordinary circumstance variation.
4.2 use the length (a) and the width (b) of vernier caliper measurement tumor behind the inoculated tumour weekly, by formula (referring to Sawaoka H, Tsuji S, Tsujii M, et al.Cyclooxygenase inhibitors sup-pressangiogenesis and reduce tumor growth in vivo[J] .Lab Invest, 1999,79 (12): 1469-77.) obtain tumor approximate volumes (V), or V=1/2ab
2, unit is cm.
4.3 adopt the disconnected neck of dislocation to put to death animal after the off-test, get tumor and weigh; Leave and take the part tumor tissue with 10% neutral formalin fix, standby.
4.4 calculate tumour inhibiting rate (%)
4.5 carry out the evaluation of medication combined effect, two medicine interaction indexes (CDI) are pressed formula and are calculated:
(two medicines have synergism when CDI＜1; CDI＜0.7 o'clock, synergism is remarkable).
5. statistical procedures: (x ± s) represent, adopts the SPSS11.5 statistical software, and measurement data adopts the t check, and enumeration data adopts variance analysis, is that difference all has significance with P＜0.05 by form with mean ± standard deviation for experimental data.
Two, experimental result
1.SPG-Rg3 inhibitory action to the growth of A549 adenocarcinoma of lung
The SPG-Rg3 medication is respectively organized the lotus tumor weight and all is starkly lower than the blank group, and three groups of its tumour inhibiting rates are all (see Table 1 and Fig. 4) more than 30%.SPG-Rg3 is after experiment the 3rd week (second week of administration), and tangible tumor-inhibiting action promptly appears in middle dosage and heavy dose of group, makes growth of tumor be considerably slower than blank group (seeing table 2 and Fig. 1 for details).The result shows that SPG-Rg3 can obviously suppress the growth of A549 adenocarcinoma of lung, and the action effect difference between each dosage group does not have significance (P〉0.05), illustrates that the tumor-inhibiting action of SPG-Rg3 does not have tangible dose dependent.SPG-Rg3 and cyclophosphamide combined medication group and simple ring phosphamide group effect no significant difference (P〉0.05), two medicine interaction indexes (CDI)〉and 1, SPG-Rg3 and the cyclophosphamide no obvious synergistic function aspect inhibition A549 adenocarcinoma of lung is described.
Table 1.SPG-Rg3 is to the inhibitory action of A549 adenocarcinoma of lung growth
The P value compares for each group and blank group;
▲Compare P between each dosage group〉0.05;
*The P value is to compare with positive controls
Table 2.SPG-Rg3 is to A549 adenocarcinoma of lung tumor volume (cm
3) influence (x ± s, n=8)
*P value＜0.05,
*P＜0.01,
* *P＜0.001, each medication group different vaccination time and corresponding blank group are relatively
2.SPG-Rg3 inhibitory action to the growth of NCI-H460 maxicell pulmonary carcinoma
The SPG-Rg3 medication is respectively organized the lotus tumor weight and is starkly lower than the blank group, and three dosage groups of its tumour inhibiting rate medication are all more than 50%.(see Table 3 and Fig. 5).The growth of NCI-H460 maxicell pulmonary carcinoma lotus tumor is faster than A549 adenocarcinoma of lung lotus tumor, and SPG-Rg3 is after experiment the 3rd week (second week of administration), and tangible tumor-inhibiting action promptly appears in middle dosage and heavy dose of group, and more obvious with the heavy dose effect.Make tumor growth be considerably slower than blank group (table 4 and Fig. 2).The result shows that SPG-Rg3 has the effect of obvious inhibition NCI-H460 maxicell pulmonary carcinoma growth, and heavy dose of group action effect is apparently higher than middle dosage group (P＜0.01).SPG-Rg3 and cyclophosphamide combined are used has Synergistic trend, but not statistically significant (P〉0.05), two medicine interaction indexes (CDI)〉and 1.Illustrate that SPG-Rg3 and the cyclophosphamide synergism aspect inhibition NCI-H460 maxicell pulmonary carcinoma is not remarkable.See Table 2 and Fig. 2.
Table 3.SPG-Rg3 is to the inhibitory action of NCI-H460 maxicell pulmonary carcinoma growth
The P value compares for each group and blank group;
▲Compare P between each dosage group〉0.05;
#The P value is to compare with positive controls
Table 4.SPG-Rg3 to the influence of NCI-H460 maxicell lung cancer tumor volume (x ± s, n=8)
*P value＜0.05,
*P＜0.01,
* *P＜0.001, each medication group different vaccination time and corresponding blank group are relatively.
▲P value＜0.05,
▲ ▲Compare between each medication group of Rg3 P＜0.01.
3.SPG-Rg3 inhibitory action to lung squamous cancer
The SPG-Rg3 medication is respectively organized the lotus tumor weight and all is starkly lower than the blank group, and three dosage groups of its tumour inhibiting rate are all (see Table 5 and Fig. 6) more than 30%.SPG-Rg3 is after experiment the 3rd week (second week of administration), tangible tumor-inhibiting action promptly appears in middle dosage and heavy dose of group, make growth of tumor obviously slow down (table 6 and Fig. 3) slowly, and from the result, find out, the growth of lung squamous cancer lotus tumor is similar to A546 adenocarcinoma of lung lotus tumor, tumor is obvious in inoculation growth all around, and the tumor formation rate of lung squamous cancer is lower than A549 adenocarcinoma of lung and H460 lung large cell carcinoma.The result shows that SPG-Rg3 can obviously suppress the growth of lung squamous cancer, acts on there was no significant difference (P〉0.05) between each dosage group of medication, illustrates that the tumor-inhibiting action of SPG-Rg3 does not have tangible dose dependent.SPG-Rg3 and cyclophosphamide combined are used has Synergistic trend, but not statistically significant (P〉0.05), two medicine interaction indexes (CDI)〉and 1.SPG-Rg is described
3Not remarkable with cyclophosphamide at the synergism aspect the inhibition lung squamous cancer.
Table 5.SPG-Rg3 is to the inhibitory action of lung squamous cancer
The P value compares for each group and blank group;
▲Compare P between each dosage group〉0.05;
#The P value is to compare with positive controls
Table 6.SPG-Rg3 is to LTEP-78 lung squamous cancer tumor volume (cm
3) influence (x ± s, n=5)
*P value＜0.05,
*P＜0.01,
* *P＜0.001, each medication group different vaccination time and corresponding blank group are relatively
What present embodiment related to is CD34 express and non-small cell lung cancer cell apoptogene express the influence of SPG-Rg3 to the non-small cell lung cancerous tissue
One, experiment material and experimental technique
1. main agents: CD34 monoclonal antibody and SP test kit (all purchase step in Foochow new); TUNEL apoptosis test kit (Roche Holding Ag)
2. experimental apparatus: FV500 laser confocal microscope (OLYMPUS); Paraffin slicing machine (German LEICA company, RM 2245); Immunohistochemical staining is hatched box, microscope (OLYMPUS BX51, Japan).
3. immunohistochemical staining method: the tumor tissues (H460 maxicell pulmonary carcinoma and A549 adenocarcinoma of lung) of getting blank group and SPG-Rg3 (1.0mg/kg) treatment group respectively fixing 24h in 10% neutral buffered formaldehyde, paraffin embedding, serial section (4 μ m), the SP method is all adopted in CD34 dyeing, presses test kit explanation step-by-step operation.Each organizes specimen with a collection of dyeing, replaces an anti-negative control of doing with PBS.Step is as follows:
(1) dewaxing: I dimethylbenzene 30min → II dimethylbenzene 30min → 100%I ethanol 5min → 100%II ethanol 5min → 95%I ethanol 5min → 95%II ethanol 5min → 80% ethanol 5min → washing
(2) the multiple antigen of hot repair: will cut into slices and invade 0.01M citrate buffer (pH6.0), electric furnace or microwave oven cut off the power supply after being heated to boiling, and behind the 5-10min, 1-2 time repeatedly, cooling back PBS washs 1-2 time at interval.
(3) E.C. 3.4.21.64 (20 μ g/ml) digestion paraffin section 5min, PBS washes.
(4) drip 3%H
2O
2Blocker, blocking-up 30min.PBS washes 3 times * 5min.
(5) Dropwise 5 % normal goats serum, sealing 30min.
(6) get rid of normal goats serum, drip suitably dilution (1:50-1:100) anti-4 ℃ of overnight incubation.PBS washes 3 times * 2min.
(7) drip the anti-mice IgG of biotinylated goat (or rabbit, human IgG), room temperature 20min, PBS washs 2 * 3min.
(8) drip SABC (Streptavidin-avidin-biotin complex) 20min PBS washing 4 * 5min.
(9) DAB colour reagent box is used in DAB colour developing.Get the 1ml distilled water, add A in the test kit, B, each 1 of C reagent adds to section, color development at room temperature, controlling reaction time under the mirror, distilled water cessation reaction behind the mixing.
(10) half oxidation haematoxylins are redyed, the hydrochloride alcohol differentiation, and the flowing water flushing is returned blue 1 minute, gradient alcohol dehydration, dimethylbenzene is transparent, neutral gum mounting, multi-functional microscopic examination and images acquired.
4. apoptosis of tumor cells detects step:
(1) at room temperature slice, thin piece is immersed the dewaxing of dimethylbenzene 30min tissue.Repeat once.
(2) in order slice, thin piece is immersed in 100%I ethanol 5min → 100%II ethanol 5min → 95%I ethanol 5min → 95%II ethanol 5min → 80% ethanol 5min → 0.85%NaCl5min under the room temperature.
(3) at room temperature immerse slice, thin piece to PBS5min flushing sample.
(4) at room temperature with slice, thin piece 15min fixing organization blocks in the 4% free methanol formalin.
(5) at room temperature immerse slice, thin piece to PBS5min flushing sample.Repeat once.
(6) remove liquid in the tissue, slice, thin piece is set level,, hatch 15min under the room temperature with E.C. 3.4.21.64 (20ug/ml).
(7) at room temperature immerse slice, thin piece to PBS5min flushing sample.
(8) at room temperature with slice, thin piece to 4% free methanol formalin 5min fixing organization blocks in PBS.
(9) at room temperature immerse slice, thin piece to PBS5min flushing sample.Repeat once.
(10) remove excess liquid on the slice, thin piece, cover tissue with 100 microlitre balance liquids.Balance 5-10min at room temperature.
(11) when structural equation, dissolve mixture of ribonucleotides on ice.For competent rTdT culture fluid is prepared in all experiments.
(12) drip Incubating Solution organizationally, cover tissue with vinyl cover, lucifuge is hatched slice, thin piece at 37 ℃ of 1h.
(13) remove vinyl cover, at room temperature slice, thin piece is immersed in cessation reaction in the dye vat of 2 * SSC.
(14) at room temperature immerse slice, thin piece to PBS5min flushing sample.Repeat secondary.
(15) PI redyes 5min, and PBS washs 3 * 5min
(16) glycerol mounting is observed under laser confocal microscope.
4. the result judges:
4.1 CD34 criterion: CD34 labelling blood capillary, the positive cell endochylema is pale brown color, calculates the blood capillary and the tiny blood vessels of tumor intrinsic color and counts tumor microvessel density (MVD).According to Weidner (referring to WeidnerN, Semple JP, Welch WR, et al.Tumor angiogenesis andmetastasis:correlation in invasivebreast carcinoma[J] .New Eng J Med, 1991,324 (1): judgment criteria 1-8), all single endotheliocyte of pale brown color or endotheliocytes of presenting are bunch all as 1 blood vessel meter, but the flesh layer is thicker and the tube chamber area is not counted greater than the blood vessel of 8 red blood cell diameters.Method of counting is at first used whole section of low power lens (100 *) pan, determines 3 visuals field (new vessels hot zone) that blood capillary is the most intensive, then in all painted blood capillaries of high power lens (400 *) field range inside counting.Get the microvessel count of the mean of 3 visual field count results for this section.
4.2 the TUNEL coloration result is observed: under the fluorescence microscope take pictures in 5 visuals field of (400 *) picked at random, carries out total cell number and the apoptosis cell that picture is handled the every pictures of back counting with Image-ProPlus v 5.1 softwares, and calculate apoptosis rate (%).
4.3 statistical procedures
Data are used the analysis of SPSS 10.0 statistical softwares.Two groups of means adopt Studentst ' s t-est more respectively, represent the dependency of two indexes with the Spearman correlation coefficient.Experimental result is represented with x ± s.
Two, experimental result
1, SPG-Rg3 is to the influence of the CD34 expression of non-small cell lung cancerous tissue
CD34 label vascular endotheliocyte, tumor tissues all have positive expression (Fig. 7～Figure 10).Endotheliocyte is dyed pale brown color, the formation tube chamber that blood capillary has, and what have only is single endotheliocyte or endotheliocyte bunch, the MVD of borderline tumor tissue is higher than central authorities.MVD obviously is less than matched group (P＜0.01) in the SPG-Rg3 treatment group tumor tissue, sees table 7 for details.
Table 7.SPG-Rg3 is to A549 adenocarcinoma of lung and H406 maxicell lung cancer tumor tissue blood vessel density and apoptotic influence
The SPG-Rg3 group compares with the blank group,
*P＜0.001,
*P＜0.0001
2, SPG-Rg3 is to the influence of non-small cell lung cancer cell apoptogene expression
Immunofluorescence result shows that apoptotic cells shows as and occurs green particles in the nucleus; Blank group apoptosis of tumor cell is rare or lose, and SPG-Rg3 treatment group shows that (Fig. 4～6) appear in more apoptotic cell, apoptosis rate (sees Table 7 and Figure 11-A～Figure 11-D), illustrate that SPG-Rg3 can promote the apoptosis of tumor cell apparently higher than the blank group.
20 (S)-ginsenoside Rg3s can obviously suppress the growth of nonsmall-cell lung cancer, and its mechanism of action is relevant with its promotion apoptosis of tumor cells and inhibition tumor-blood-vessel growth, and is not obvious with the synergistic function that cyclophosphamide combined is used.The effect that 20 (S)-ginsenoside Rg3s can obviously suppress the nonsmall-cell lung cancer growth belongs to discovery first, for the medicine that 20 (S)-ginsenoside Rg3s is developed to a kind of people's of treatment nonsmall-cell lung cancer provides foundation fully.
Claims (1)
1.20 (S)-ginsenoside Rg3 is as the application in the medicine of preparation treatment LTEP-78 lung squamous cancer type nonsmall-cell lung cancer.
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| CN102423316B (en) * | 2011-12-31 | 2013-08-21 | 中国人民解放军军事医学科学院野战输血研究所 | Paclitaxel compound preparation and preparation method thereof |
| CN102626416B (en) * | 2012-05-11 | 2013-04-17 | 江苏省人民医院 | Application of tamoxifen, rapamycin and ginsenoside Rg3 composition in preparing medicaments for treating liver cancer |
| CN104644661A (en) * | 2013-11-21 | 2015-05-27 | 富力 | Application of 20(R)-ginsenoside Rg3 in preparation of medicament for relieving or/and treating hepatitis B and medicament |
| CN105777838A (en) * | 2014-12-17 | 2016-07-20 | 富力 | 20(R)-ginsenoside Rg3 derivatives, and preparation method and application thereof |
| CN107929737A (en) * | 2018-01-15 | 2018-04-20 | 福州大学 | A kind of pharmaceutical composition of anti-tumor metastasis and its application |
| CN110960540B (en) * | 2018-09-29 | 2021-11-09 | 中国医学科学院药物研究所 | Use of 3 beta-O-Glc-DM and 20S-O-Glc-DM for treating lung or colorectal cancer |
| CN113082039B (en) * | 2021-03-30 | 2023-01-13 | 香港浸会大学深圳研究院 | Composition for treating sorafenib drug-resistant tumor and application thereof |
| CN114903909A (en) * | 2022-06-09 | 2022-08-16 | 云南大唐汉方制药股份有限公司 | Preparation and application of combined use of artemisinin and ginsenoside Rg3 in adjuvant therapy or therapy of primary lung cancer |
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