CN109828111A - Purposes of the MFAP5 in the preparation that preparation screening alpha interferon intervenes liver cancer sensitive group marker - Google Patents
Purposes of the MFAP5 in the preparation that preparation screening alpha interferon intervenes liver cancer sensitive group marker Download PDFInfo
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- CN109828111A CN109828111A CN201711200417.2A CN201711200417A CN109828111A CN 109828111 A CN109828111 A CN 109828111A CN 201711200417 A CN201711200417 A CN 201711200417A CN 109828111 A CN109828111 A CN 109828111A
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- liver cancer
- mfap5
- alpha interferon
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- sensitive group
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Abstract
The invention belongs to biomedical clinical detection technique fields, and in particular to purposes of the MFAP5 in the preparation that preparation screening alpha interferon intervenes liver cancer sensitive group marker.The present invention tests and analyzes alpha interferon intervention with the liver cancer patient tissue samples that do not intervene using the method for RNA-seq high throughput express spectra sequencing, filter out the secretory protein MFAP5 of differential expression, with immunohistochemistry, tri- kinds of methods of Western blot and ELISA are identified, prove that the MFAP5 secretory protein has significant differential expression in alpha interferon intervention and the human liver cancer tissue that do not intervene, there are obvious differential expressions in human peripheric venous blood simultaneously, the MFAP5 is the key molecule during alpha interferon intervention liver cancer, it can be used as the marker that screening alpha interferon intervenes liver cancer sensitive group, it is further used for screening alpha interferon intervention treatment liver cancer sensitive group, and accuracy is high.
Description
Technical field
The invention belongs to biomedical clinical detection technique fields, are related to the new medical application of MFAP5 albumen, specifically relate to
And purposes of the MFAP5 albumen as detection marker in the preparation for being used to prepare screening alpha interferon intervention liver cancer sensitive group.
Background technique
Data discloses primary carcinoma of liver (Hepatocellular carcinoma, HCC) and occupies global Incidence
Rate the 6th, death rate second is the second largest tumor lethal reason in China.By the effort of decades, China's liver cancer research
Remarkable break-throughs are had been achieved with, but the postoperative 5 years transfer and relapse rates of its radical excision are still greater than 60%.Postoperative recurrence shifts
Further improve the major obstacle of liver cancer patient prognosis.It is useful clinically so far effectively to inhibit liver cancer recurrence and metastasis after resection
Drug be still rarely seen.
Prior art discloses alpha interferons as a multiduty cell factor, is listed in chronic hepatitis B always
Fiest-tire medication.Research in recent years gradually finds that it has the work for inhibiting tumour growth and transfer and relapse in entity tumor
With.In numerous anticancer drugs, alpha interferon is a small number of to one of effective drug of liver cancer, has huge clinical value；
There is studies have shown that difference lotus knurl individual to have differences drug susceptibility, causes alpha interferon to the general curative effect mesh of liver cancer patient
Before can't fully meet clinical requirement, think that can its curative effect further increase in the industry and depend on the effective identification of searching
The molecular marker of sensitive group.
It is a kind of multifunctional protein prior art discloses MFAP5, assembles and adjust endothelial cell row in elastic microfibers
It plays an important role in, it is considered to be the New Regulator of cell survival, however effect of the MFAP5 in cancer still needs to be explained
It is bright.Alpha interferon is widely used in clinical anti-liver cancer and anti-transfer and relapse treatment at present, still, so far, there is not yet about MFAP5
Intervene the report of the marker of liver cancer sensitive group as screening alpha interferon.
Status based on the prior art, present inventor is quasi- to provide the new medical application of MFAP5 albumen, specifically relates to
And purposes of the MFAP5 albumen as detection marker in the preparation for being used to prepare screening alpha interferon intervention liver cancer sensitive group.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, providing the new medical application of MFAP5 albumen, specifically relate to
And purposes of the MFAP5 albumen as detection marker in the preparation for being used to prepare screening alpha interferon intervention liver cancer sensitive group.
The present invention provides MFAP5 albumen or its specific antibody, and detection marker to be used as to be used to prepare screening α interference
Element intervenes the purposes in the preparation of liver cancer sensitive group.
The present invention is using the method for RNA-seq high throughput express spectra sequencing to alpha interferon intervention and the human liver cancer that do not intervene
Tissue tested and analyzed, filter out the secretory protein MFAP5 of differential expression, with immunohistochemistry, western blot and
Tri- kinds of methods of ELISA are identified, it was demonstrated that the albumen has significant table in alpha interferon intervention and the human liver cancer tissue that do not intervene
Up to difference, the mRNA and protein level of MFAP5 significant raising in the patient of prognosis mala, while being deposited in human peripheric venous blood
In notable difference, the MFAP5 albumen or its specific antibody can be used as the key molecule during alpha interferon intervention liver cancer
And the marker as diagnosis interferon alfa liver cancer sensitive group, it is further used for preparation screening alpha interferon and intervenes liver cancer
The preparation of sensitive group, and the accuracy diagnosed is high.
The further object of the present invention is to provide a kind of examination for intervening liver cancer sensitive group for detecting screening alpha interferon
Agent box.
To realize above-mentioned further object, the technical solution adopted by the present invention is that:
A kind of kit for intervening liver cancer sensitive group for detecting screening alpha interferon, the kit include detection
The reagent of MFAP5 protein content.
In the embodiment of the present invention, the kit (10 that liver cancer sensitive group is intervened for detecting screening alpha interferon
Person-portion) there are following components to be made: 2.5ug/ml humanized's soluble M FAP5 antibody, the sheep anti-mouse igg of HRP label, DAB colour developing
Liquid (Wuhan doctor's moral company), haematoxylin redyes liquid and 3%H2O2.In the embodiment of the present invention, using kit pair obtained
Liver cancer tissue and cancer beside organism carry out laboratory test results and show, according to dye levels+(< 20%), ++ (20-50%), +++
(51-75%), +++ 4 grades of+(76-100%) scores, and analyzes the results show that MFAP5 positive rate reaches in liver cancer
41.81%, have significant difference (as shown in Figure 1)；Liver cancer is carried out using Western blot in the embodiment of the present invention to face
The detection of bed sample, the results show that the MFAP5 positive sample of identification meets Western blot testing result (as shown in Figure 2)；
In the embodiment of the present invention, the detection of liver cancer clinical sample, the MFAP5 positive sample identified as the result is shown are carried out using ELISA
Meet Western blot testing result (as shown in Figure 3)；In the embodiment of the present invention, by the liver cancer patient of collection and normal disease
Human blood sample, is tested using ELISA, the results show that the mRNA and protein level of MFAP5 are outside liver cancer patient and normal patient
There are notable differences in all venous blood；In the embodiment of the present invention, prognosis that data obtained and hospital are provided, recurrence rate
Shown after comparing, MFAP5 positive patient shows treatment poor prognosis, high recurrence rate as a result, therefore, the MFAP5
It can be used as the judgment criteria of prognosis in hcc recurrence, have significant difference (as shown in Figure 4).
The present invention is further prepared for a kind of reagent that can be detected screening alpha interferon and intervene the marker of liver cancer sensitive group
Box provides clinical reference foundation for the MFAP5 screening for intervening liver cancer sensitive group for liver cancer alpha interferon.
Compared with prior art, MFAP5 kit of the present invention has the following advantages that and conspicuousness progress:
1, sensibility: kit sensibility of the invention is high, the MFAP5 antibody and prior art others liver cancer mark
Object is compared, and positive strength can achieve ++++, meet clinical monitoring standard.
2, accuracy: kit accuracy of the invention is high, MFAP5 antibody and prior art others liver cancer marker
It compares, accuracy rate is higher, meets clinical monitoring standard.
Detailed description of the invention
Fig. 1 present invention is used for the picture (20X) of liver cancer patient immunohistochemical experiment: graphical results show that MFAP5 is dry in α
It disturbs to express in element intervention liver cancer patient and be positive；It expresses and is negative in interferon alfa liver cancer patient cancer side.
Fig. 2 Western blot verifies expression of the MFAP5 in the liver cancer patient cancerous tissue and cancer beside organism, as a result
Display is consistent with the result of Fig. 1.
Fig. 3 ELISA verifies expression statistical chart of the MFAP5 in liver cancer patient blood serum, as the result is shown in liver cancer patient
The expression of MFAP5 has apparent difference, P < 0.001 compared with Healthy People in serum；As the result is shown with the result phase of Fig. 1 and Fig. 2
Meet.
Fig. 4 liver cancer patient MFAP5 positive and prognosis, the relationship of recurrence: the liver cancer patient filtered out according to Examples 1 and 2
MFAP5 positive patient, by finding after comparing with treatment prognostic data, the prognosis after interferon therapy of MFAP5 positive patient is good,
Recurrence rate is low, has significant difference, shows that MFAP5 can be used as the mark of interferon alfa liver cancer patient sensitive group screening
Will object.
It elaborates below with reference to embodiment and attached drawing to the present invention, but implementation of the invention is not limited to that.
Specific embodiment
What the present invention used is able to detect the kit of human peripheric venous blood MFAP5 expression quantity, by humanized's MFAP5 antigen
Antibody, the sheep anti-mouse igg of HRP (horseradish peroxidase) label, DAB developing solution and haematoxylin redye liquid composition.
1 kit forms of embodiment:
Liver cancer interferon alfa sensitive group Screening Diagnosis kit (10 person-portion)
[(1) 2.5ug/ml humanized's soluble M FAP5 antibody 0.5ml
(2) the sheep anti-mouse igg 0.5ml of HRP label
(3) DAB developing solution (Wuhan doctor's moral company) 0.5ml
(4) haematoxylin redyes liquid 0.5ml
(5) 3%H2O2 0.5ml
The application method of kit:
(1) roasting piece: 91.7 DEG C, 30min；It is placed in dimethylbenzene I after taking-up；
(2) it dewaxes: being successively placed in each 10min of dimethylbenzene I and II；
(3) alcohol aquation: successively it is placed in alcohol (100%, 95%, 80%, 75%) each 5min of various concentration；
(4) it is developed a film with PBS, 5min, 3 times.Deactivating endogenous peroxydase: every gives 200ul H2O2 (6%), sets
In wet box 15min.It is developed a film with PBS, 5min, 3 times；
(5) hot repair is multiple: repairing in 1 × EDTA and is placed in slice in liquid.It boils, the low fire 10min of micro-wave oven, after cooling 3min,
Low fire 7min again.Slice antigen gives 5%BSA closing, is put into wet box 20min, reject BSA, indicates to be added by antibody specification
Primary antibody.It 4 DEG C, is put into wet box and is incubated for 18hrs, developed a film with PBS, 5min, 3 times；
(6) it under room temperature, is added in A liquid wet box and is incubated for 30min, developed a film again with PBS, 5min 3 times, is placed in DAB working solution
Afterwards, it observes under the microscope at once, is added in PBS in due course and terminates reaction, after haematoxylin redyeing, shake up, PBS is rinsed.Add HCl de-
Color；
(7) 6.1 dehydrations: alcohol at different levels, concentration (75%, 80%, %%, 100%) each 5min from low to high；Dimethylbenzene is each
5min。
(8) 6.1 mountings: taking out slice in dimethylbenzene, air-dry, add a little neutral gum, give clean coverslip mounting.
Embodiment 2
The detection of kit progress liver cancer clinical sample
Liver cancer clinical samples 170, including liver cancer tissue and cancer beside organism (as shown in Figure 1) are had collected, according to kit
Operation instruction scheme is tested, the results show that according to dye levels+(< 20%), ++ (20-50%), +++ (51-75%),
++++(76-100%) 4 grades score, finally analyzed with statistical software, MFAP5 positive rate reaches in liver cancer
41.81%, there is significant difference.
Embodiment 3
The detection of Western blot progress liver cancer clinical sample
By 10 pairs of operation of liver cancer samples of collection, Western blot is tested, and is identified in embodiment 2 as the result is shown
MFAP5 positive sample meets Western blot testing result (as shown in Figure 2).
Embodiment 4
The detection of ELISA progress liver cancer clinical sample
It by 10 liver cancer patients and normal patient blood preparation each 5 of collection, is tested using ELISA, the results show that real
It applies the MFAP5 positive sample identified in example 2 and meets Western blot testing result (as shown in Figure 3).
Embodiment 5
According to the prognosis that embodiment 2 and 3 the data obtained of embodiment and hospital provide, found after recurrence rate comparing,
There is treatment poor prognosis in MFAP5 positive patient, and high recurrence rate has significant difference (as shown in Figure 4), therefore MFAP5 can be with
Judgment criteria reference mark as prognosis in hcc recurrence.
The present invention is prepared for a kind of mark for being able to detect screening interferon alfa liver cancer sensitive group through experimental study
The kit of object provides clinical reference foundation in the screening of liver cancer interferon alfa liver cancer sensitive group for MFAP5, and existing
Technology is compared, and MFAP5 kit of the present invention has the following advantages that and conspicuousness progress:
1, sensibility: kit sensibility of the invention is high, the MFAP5 antibody and other liver cancer markers on the market
Compare, positive strength can achieve ++++, meet clinical monitoring standard；
2, accuracy: kit accuracy of the invention is high, MFAP5 antibody and other liver cancer marker phases on the market
Than accuracy rate is higher, meets clinical monitoring standard.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (5)
- Purposes of the 1.MFAP5 in the preparation that preparation screening alpha interferon intervenes liver cancer sensitive group marker.
- 2. purposes according to claim 1, which is characterized in that the MFAP5 is in alpha interferon intervention and the people liver that do not intervene There is significant differential expression in cancerous tissue.
- 3. purposes as described in claim 2, which is characterized in that MFAP5 masculine liver cancer patient's prognosis is significantly worse than PAPSS1 negative patient；MFAP5 negative HCC patient can be obviously improved patient's prognosis after alpha interferon is intervened, and have conspicuousness Difference.
- 4. a kind of kit for intervening liver cancer sensitive group for Testing and appraisal alpha interferon, which is characterized in that the kit Reagent comprising detecting MFAP5 protein content.
- 5. kit according to claim 4, which is characterized in that the kit is soluble by 2.5ug/ml humanized The sheep anti-mouse igg of PAPSS1 antibody, HRP label, DAB developing solution, haematoxylin redye liquid, 3%H2O2Composition.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148547A (en) * | 2009-07-13 | 2016-11-23 | 霍夫曼-拉罗奇有限公司 | Diagnostic method and composition for treatment of cancer |
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- 2017-11-23 CN CN201711200417.2A patent/CN109828111A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148547A (en) * | 2009-07-13 | 2016-11-23 | 霍夫曼-拉罗奇有限公司 | Diagnostic method and composition for treatment of cancer |
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