CN115708833A - Application of gemcitabine in preparation of anti-hydatid medicament - Google Patents
Application of gemcitabine in preparation of anti-hydatid medicament Download PDFInfo
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Abstract
The application of gemcitabine in preparation of the anti-echinococcus hepatica drug can influence the DNA synthesis of echinococcus granulosus and echinococcus multilocularis to cause the echinococcus granulosus and echinococcus multilocularis to undergo apoptosis, and the alkaline phosphatase test and the trypan blue staining test verify that the gemcitabine can effectively kill the echinococcus granulosus and echinococcus multilocularis, so that the gemcitabine can effectively kill the echinococcus granulosus and echinococcus multilocularis in vitro, and can be used as a drug or a drug component for mainly or assisting in killing the echinococcus hepatica, and a reference is provided for developing the anti-echinococcus drug replacing albendazole.
Description
Technical Field
The invention relates to the field of anti-echinococcosis drugs, and in particular relates to application of gemcitabine in preparation of an anti-echinococcosis liver drug.
Background
Echinococcosis, also known as echinococcosis, is a parasitic disease of zoonosis caused by the parasitosis of echinococcus larvae in human or animal bodies. The developed areas of animal husbandry such as Tibet, xinjiang, ningxia, gansu, neimao and Qinghai in China are epidemic areas of echinococcosis, and the threatened population is nearly 6600 ten thousand. Infection by echinococcosis can cause damage to organs and tissues such as liver, lung, brain and bones, and the patients lose labor capacity, which is one of the main causes of disease-induced poverty and disease-induced poverty of the masses in part of farming and pastoral areas. Meanwhile, echinococcosis also has the characteristics of infectivity, latency and difficult detection of early infection, and if the echinococcosis is incompletely killed, the echinococcosis can bring a heavy burden of 'poverty caused by diseases' to the public in an epidemic area.
Echinococcosis in china mainly includes Cystic Echinococcosis (CE) caused by parasitizing Echinococcosis granulosa (Eg) larvae with human tissue and organs, and Alveolar Echinococcosis (AE) caused by parasitizing Echinococcosis granulosa (Em) larvae with cyst larvae with Echinococcosis multilocularis (Em). If the patient with alveolar echinococcosis is not treated, the 10-year mortality rate can reach 94 percent, which is also called as 'insect cancer'.
In the prior art, the main medicine for clinically treating echinococcosis is albendazole. However, zhang Imamine et al, in the analysis of Albendazole-induced 38 cases of adverse reactions in children, indicate that albendazole needs to be taken for a long time and has a large adverse reaction, and do not suggest that albendazole be used for treating echinococcosis. Zhengrongyuan et al also pointed out the same problem in the report on reevaluation of safety after albendazole was marketed. Therefore, there is a need to find other suitable drugs that can inhibit the growth of hydatid.
Disclosure of Invention
The invention aims to provide an application of gemcitabine in preparation of anti-echinococcus hepatica drugs, which is verified by an alkaline phosphatase test that gemcitabine can effectively kill echinococcus granulosus, and trypan blue staining is utilized to evaluate the killing effect of gemcitabine on primary echinococcus granulosus segments, so that gemcitabine can effectively kill primary echinococcus granulosus segments and echinococcus polyspora, and can be possibly used as an anti-echinococcus drug, and the problems that albendazole needs to be taken for a long time, adverse reactions are large, and the cure rate is low in the prior art are solved.
Specifically, the purpose is realized by the following technical scheme:
application of gemcitabine in preparing medicine for resisting liver infusorian.
Gemcitabine is a cytosine nucleoside derivative, often present as the hydrochloride salt in pharmaceuticals. At present, gemcitabine is mainly used as a second-line drug after fluorouracil fails in patients with advanced pancreatic cancer, and can improve the quality of life of the patients; secondly, it is used as the first line for the locally advanced (stage III) and non-small cell lung cancer with metastasis (stage IV). It has been shown that gemcitabine has palliative therapeutic effects on ovarian cancer, breast cancer, bladder cancer, cervical cancer, liver cancer, biliary tract cancer, nasopharyngeal cancer, testicular tumor, lymphoma, mesothelioma, and head and neck cancer.
However, the application of gemcitabine in the preparation of anti-hydatid drugs is still blank. The inventor proves that the gemcitabine has the effect of killing echinococcus granulosus and echinococcus multocida in vitro through experiments. Through the alkaline phosphatase test, the concentration of the alkaline phosphatase in the culture supernatant is obviously increased after gemcitabine acts for 36 hours in the gemcitabine experimental group compared with the control group and the albendazole experimental group adopted in the prior art, which indicates that more echinococcus multilocularis is killed, so that the alkaline phosphatase in the cyst wall is released into the culture supernatant. Meanwhile, the killing effect of the drug on echinococcus granulosus protocephalic nodules is analyzed through trypan blue staining, the survival rate of the echinococcus granulosus after gemcitabine treatment for 24 hours is less than 20%, the survival rate of the echinococcus granulosus after 48 hours is basically close to 0%, and the killing efficiency is far higher than that of an albendazole experimental group. Therefore, the gemcitabine can effectively kill echinococcus granulosus and echinococcus multocida in vitro, and can be used as a medicine or a medicine component for mainly or auxiliarily killing the hydatid.
Further, the route by which gemcitabine kills echinococcus granulosus and echinococcus multocida may be that it acts upon activation by deoxycytidine kinase after entry into the parasite to form gemcitabine phosphate (dFdCMP), gemcitabine diphosphate (dFdCDP), and gemcitabine triphosphate (dFdCTP), where dFdCDP and dFdCTP are active species that inhibit DNA synthesis; the dFdCDP reduces the production of deoxynucleoside triphosphates (necessary for DNA synthesis), particularly deoxycytidine triphosphate (dCTP), by inhibiting RNA reductase, which ultimately leads to apoptosis; dFdCTP competes with dCTP for incorporation into the DNA strand (self-enhancement) to elongate the DNA strand, and the incorporated dFdCTP cannot be removed by DNA polymerase, so that the extended DNA strand cannot be repaired, thereby inhibiting DNA synthesis and finally causing apoptosis.
Further, the medicine also comprises pharmaceutically acceptable auxiliary materials. The adjuvant can be at least one of starch, sugar powder, and glycerol.
Further, the dosage form of the medicine is injection.
In summary, the invention has the following advantages and beneficial effects: gemcitabine can cause apoptosis of echinococcus granulosus and echinococcus multilocularis by influencing DNA synthesis of the echinococcus granulosus and echinococcus multilocularis, and alkaline phosphatase test and trypan blue staining test verify that gemcitabine can effectively kill the echinococcus granulosus and echinococcus multilocularis, so that gemcitabine can effectively kill the echinococcus granulosus and echinococcus multilocularis in vitro, and can be used as a drug or a drug component for mainly or assisting killing of periphyton hepatica, and a reference is provided for developing anti-echinococcosis drugs replacing albendazole.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 shows the effect of alkaline phosphatase assay for killing Echinococcus multiplex in Gemcitabine (Gemcitabine), albendazole (Albendazole) and control (DMSO, 0.2% dimethylsulfoxide) in the alkaline phosphatase assay in a specific example of the invention, where the abscissa is treatment time and the ordinate is absorbance A 405 A value;
FIG. 2 is a graph showing the effect of trypan blue staining on killing of Echinococcus granulosus protopanoxas in gemcitabine, albendazole, and control in an embodiment of the present invention, wherein the abscissa is the treatment time and the ordinate is the survival rate of the protopanaxas.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
The technology related to the invention is a conventional technical means of molecular cloning, wherein related enzymes, primers, reagents and reaction conditions can be reasonably selected according to the experience of a person skilled in the art without description, related reagent consumables belong to common products sold in the market, and related detection means and instruments are all well known and are well mastered by the person skilled in the art.
In the present invention, the data was analyzed using Prism version 9 (GraphPad Software) statistical Software in the examples.
[ example 1 ]
The killing effect of the medicine on the echinococcus multilocularis is detected by an alkaline phosphatase experiment.
The alkaline phosphatase test can detect the content of alkaline phosphatase released into the culture solution supernatant after the bladder wall of the echinococcus multilocularis is damaged, so as to evaluate the effect of the medicine on the echinococcus multilocularis.
(1) Experimental Material
The echinococcus multicavis is obtained from abdominal cavity of Kandelia parviflora infected with echinococcus multicavis, the feeder cells are human liver cancer cell strain QGY-7701, and the culture medium is DMEM culture medium.
(2) Experimental method
Washing Echinococcus multilocularis twice with Hanks solution, placing Echinococcus multilocularis in culture flask coated with human liver cancer cell QGY-7701, adding DMEM culture medium, and 5% 2 After 2 months of culture at 37 ℃, vesicles with the diameter of 1-5 mm are collected. Washed with Hanks solution, resuspended in DMEM medium, and added to 24-well culture plates, each well containing 2ml of 25-35 vesicles.
The experimental groups included the gemcitabine group, the albendazole group, and the 0.2% dmso control group. Each drug was provided with 2 parallel wells at a final concentration of 40. Mu. Mol/L, repeated 2 times. 5% of CO 2 After incubation at 37 ℃ for 36 hours and 120 hours, 170. Mu.L of alkaline phosphatase reaction solution was added to each well of a 96-well plate, 30. Mu.L of the supernatant was added thereto and mixed, and the mixture was incubated at 37 ℃ for 30 minutes and then the absorbance A was measured 405 The value is obtained.
(3) Results of the experiment
As shown in FIG. 1, after 36 hours of treatment, the absorbance values of the gemcitabine group were significantly higher than those of the albendazole group and the control group, and the culture supernatants of the gemcitabine group, the albendazole group and the control groupA of alkaline phosphatase 405 The values are 0.524 + -0.052, 0.246 + -0.019 and 0.115 + -0.009, respectively. A of alkaline phosphatase in supernatant of Gemcitabine group after 120 hours of action 405 The value is as high as 0.608 +/-0.072, while the albendazole group and the control group are respectively 0.424 +/-0.038 and 0.116 +/-0.010. It can be seen that gemcitabine kills echinococcus multilocularis more strongly than albendazole after 36 hours and 120 hours of treatment.
[ example 2 ]
Trypan blue staining was used to analyze the killing effect of the drug on echinococcus granulosus protocoid.
Trypan blue can be used to stain and count surviving protoheads, and then analyze the viability of protoheads over the incubation period. The initial survival rate of the procephalic segment is greater than 90%.
(1) Experimental Material
Echinococcus granulosus is extracted from cyst fluid of naturally infected echinococcus granulosus-infected sheep liver vesicles in RPMI 1640 medium containing 25mmol/L Hepes, 2mmol/L L-glutamic acid, 100U/mL penicillin, and 100. Mu.g/mL streptomycin.
(2) Experimental methods
Standing the cyst fluid to naturally settle the echinococcus granulosus protocephalus, washing with Hanks fluid for 3 times, then re-suspending, taking 200 mu L of re-suspension, staining with 0.4% trypan blue and counting the live protocephalus.
Procephalic nodules were incubated with RPMI 1640 and 1mL of culture suspension was added to each well of a 24-well plate, with approximately 100 procephalic nodules.
The experimental groups included the gemcitabine group, the albendazole group, and the 0.2% dmso control group. Each drug was provided with 2 parallel wells at a final concentration of 40. Mu. Mol/L, repeated 2 times. 5% of CO 2 After incubation at 37 ℃ for 1, 2, 3, 4, 5, 6 and 7 days, viability of the protocephalic segments was calculated by staining with 0.4% trypan blue and calculating the viable protocephalic segments.
(3) Results of the experiment
As shown in FIG. 2, the survival rate of echinococcus granulosus after 1 day of gemcitabine treatment was 18.33. + -. 4.509%, the survival rate of echinococcus granulosus after 2 days was 1.333. + -. 1.528%, and all of the original head segments of the gemcitabine group died at day 3. The survival rate of the protocephalad of the albendazole group is gradually reduced within one week, and 6.667 +/-2.517% of the protocephalad still survives by the 7 th day. It can be seen that gemcitabine kills echinococcosis more effectively than albendazole.
In conclusion, the in vitro killing effect of gemcitabine on echinococcus multocida and echinococcus granulosus is obviously better than that of albendazole, and meanwhile, gemcitabine is used for a human body as a mature anti-cancer drug, so that gemcitabine is expected to be used as a substitute for albendazole to be used as a drug or a drug component for mainly or auxiliarily killing hydatid cochleariae.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. Application of gemcitabine in preparing medicine for resisting liver infusorian.
2. Use according to claim 1, characterized in that the medicament is for killing echinococcus granulosus and/or echinococcus multocida.
3. The use according to claim 2, wherein the medicament is for affecting the DNA synthesis of echinococcus granulosus and/or echinococcus multilocularis, resulting in apoptosis of the echinococcus granulosus and/or echinococcus multilocularis.
4. Use according to claim 3, wherein the medicament is for forming gemcitabine diphosphate and gemcitabine triphosphate for inhibiting DNA synthesis of echinococcus granulosus and/or echinococcus multocida.
5. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.
6. The use according to claim 1, wherein the medicament is in the form of an injection.
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Non-Patent Citations (1)
| Title |
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| 王帅: "牛带绦虫和亚洲带绦虫全基因组测序、比较基因组及胰岛素信号通路研究", 《中国博士学位论文全文数据库 农业科技辑》, no. 1, pages 050 - 82 * |
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