CN108815186A - A kind of drug and preparation method and application treated HPV persistent infection and cause disease - Google Patents
A kind of drug and preparation method and application treated HPV persistent infection and cause disease Download PDFInfo
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Abstract
The invention belongs to biotechnology and its clinically applied technical field, present invention firstly discovers that, the supernatant suspension obtained using immune cell expansion is that main pharmacodynamics ingredient is applied to uterus neck, treats HPV infection or the disease due to caused by HPV persistent infection.By the positive patient for the HPV infection that the method is treated, after 7-8 treatment, negative conversion rate reaches 30% or more, and negative conversion rate can reach 50% or more after 15 to 20 treatments, and can be effectively controlled and treat the change of uterine neck tumor sample caused by HPV（CIN）And cervical cancer relapse or transfer, achieve the effect that prevent and treat cervical carcinoma.So far, the method for making HPV persistent infection quickly turn out cloudy and prevent and treat cervical carcinoma is smeared by immunocyte correlation external application both at home and abroad without report, the method is pioneering both at home and abroad.
Description
Technical field
The invention belongs to biotechnology and its clinically applied technical field, and in particular to a kind of HPV that treats persistently feels
Contaminate the drug for causing disease and preparation method and application.
Background technique
Human papilloma virus (human papillomavirus, HPV) is a kind of papilloma for belonging to papovaviridae
Vacuolating virus A belongs to, and is spherical DNA virus, the hyperplasia of squamous epithelium of the strong-willed human skin mucous membrane of energy.Show as verruca vulgaris, sharp
The symptoms such as condyloma, with the disease incidence rising of condyloma acuminatum in venereal disease and increasing for cervical carcinoma, cancer of anus, cancer of the esophagus etc., human milk
Head tumor virus (HPV) infection increasingly causes to pay close attention to.The HPV crowd infection rate of skin-type is very universal, seeks Ru above-mentioned common
Normal wart, toe wart, verruca plana etc., the high-risk HPV to arouse attention infects uterine neck caused by the low risk HPV infection with external genital organs
Cancer and genital wart, according to statistics in global venereal disease, genital wart caused by HPV infection accounts for 15-20%.
HPV infection is the basic pathogenic factor of cervical carcinoma.HPV can usually die away after infection, only lasting height
Danger type human papilloma virus infection is only the basic reason for leading to cervical carcinoma.As a kind of common sexually transmitted disease, uterine neck sense
Dye high-risk HPV be it is very universal, HPV infection is mostly transience, has the patient of 10%-15% in persistent infection shape
State.Many is that the necessary condition of cervical lesions occurs researches show that the infection of duration high-risk HPV.HPV is generally deposited in crowd
In, U.S. one, investigation display 1/3 has the collegegirl of sexual behaviour that HPV-DNA can be detected, and 70% women has No. 1 machine in life
High-risk HPV can be infected.The existing illness rate of HPV is estimated as 25%-39%, depends primarily on age group, residence and detection
Means.The peak age of genital HPV infection is that 5%-10% is high-risk HPV persistent infection state after l8-28 years old, 35 years old, is held
Cervical intraepithelial neoplasia (CIN) (cervical intraepithelial occurs for the women of continuous infection HPV high-risk-type virus
Neoplasia, CIN) risk increase 100-300 times.Persistent infection HPV high-risk-type virus is to be led to and maintain epithelium of cervix uteri weight
Spend the necessary condition of atypical hyperplasia (cervical intraepithelial neoplasia 3, CIN II) lesion.The world
The case-control study that different regions are carried out is shown：Relative risk 50-100 of the HPV infection in cervical cancer pathogenesis, and
HPV16,18 relative risks infected are more up to 100-500.It can be detected in almost all cervical carcinoma sample high-risk
The presence of group HPV, the recall rate of HPV-DNA is 70%-90% in precancerous lesion (i.e. II, III grade of intraepithelial neoplasia),
It is 20%-50% in I grades of intraepithelial neoplasia (cin)s.According to another scientific investigations showed that the generation of cancer of the esophagus reaches with the HPV infection degree of correlation
60%-80%.
Summary of the invention
In order to overcome the problems of in the prior art, the purpose of the present invention is to provide it is a kind of treat HPV infection or
It treats HPV persistent infection and causes drug of disease and the preparation method and application thereof.What the drug can be smeared directly by external application
Mode can preferably treat HPV infection or the disease due to caused by HPV persistent infection, mitigate sufferer pain.
To achieve the goals above and other related purposes, the present invention adopt the following technical scheme that：
The first aspect of the present invention, provide immune cell expansion suspension be used to prepare treatment HPV infection or HPV infection cause
Disease drug purposes.
In a kind of embodiment, the immune cell expansion suspension is main pharmacodynamics ingredient.
In a kind of embodiment, the immune cell expansion suspension refers to that immunocyte obtains after amplification cultivation, separation
Supernatant.Therefore, the immune cell expansion suspension is also referred to as：The suspension of acquisition is centrifuged after immune cell expansion.
It should be noted that being free of immunocyte in the immune cell expansion suspension.
In a kind of embodiment, when amplification cultivation, X-VIVO15 is can be used in culture medium.
In a kind of embodiment, after amplification cultivation, immunocyte density reaches 2.0 × 106A cell/mL or more.
In a kind of embodiment, after amplification cultivation, immunocyte density reaches 3.0 × 106A cell/mL or more.
In a kind of embodiment, after amplification cultivation, immunocyte density reaches 4.0 × 106A cell/mL or more.
Existing amplification method in the prior art can be used when the amplification.
In a kind of embodiment, when amplification, used method was selected from：Cell factor stimulus method, antibody stimulating method
Or trophocyte's amplification.
In a kind of embodiment, the cell factor stimulus method refers to：Using IL-2, IL-12, IL-18,4-1BB, IL-
21, the cell factors such as IL-15 carry out stimulation induced amplification.The cell mainly obtained is the cell mixing of NKT cell and T cell.
In a kind of embodiment, the antibody stimulating method refers to：Using CD16 monoclonal antibody, IL-2, IL-12, IL-21/IL-
15 equal antibody carry out induction stimulation amplification.The cell mainly obtained is still the cell mixing of NKT cell and T cell.
In a kind of embodiment, trophocyte amplification refers to：Use tumour cell as trophocyte's parent, by 4-
1BB, IL-21 or IL-15, CD86 etc. are transfected into cell membrane surface, using irradiation etc. inactivations mode handle, by a certain percentage plus
Enter in cultivating system, with IL-12, IL-18, CD16 monoclonal antibody Co stituation induced amplification, the NK cell purity of acquisition generally can be big
In 70%.The cell mixing accounting of NKT and T cell reduces.
In a kind of embodiment, the immune cell expansion suspension is that NK cell expands suspension.The NK cell amplification is outstanding
Liquid refers to the supernatant that NK cell obtains after amplification cultivation, separation.Therefore, it is thin to be also referred to as NK for the NK cell amplification suspension
The suspension of acquisition is centrifuged after born of the same parents' amplification.
It should be noted that not containing NK cell in the NK cell amplification suspension.
In a kind of embodiment, NK cell can be to be obtained by monocyte culture.
In a kind of embodiment, the cells of monocytic origin is in peripheral blood, Cord blood or placental blood etc..
In a kind of embodiment, the HPV infection refers to HPV test positive.
In a kind of embodiment, disease caused by the HPV infection refers to disease caused by HPV persistent infection.
In a kind of embodiment, disease caused by HPV persistent infection is that uterine neck tumor sample becomes (CIN), condyloma acuminatum or uterine neck
Cancer.
In a kind of embodiment, the drug is external preparation.
In a kind of embodiment, the drug is that preparation is smeared in external application.
In a kind of embodiment, the drug is exterior-applied gel.
In a kind of embodiment, the main pharmacodynamics ingredient of the external-use gel preparation is：Immune cell expansion suspension.
In a kind of embodiment, in the external-use gel preparation other than main pharmacodynamics ingredient, also containing can pharmaceutically connect
The carrier auxiliary material received.These carrier auxiliary materials can be the customary adjuvant for external-use gel preparation, such as carbomer.The use of auxiliary material
Amount, those skilled in the art can determine according to actual needs.For example, 3-5% (the quality hundred of entire external-use gel preparation can be accounted for
Divide ratio).
In a kind of embodiment, the preparation method of the external-use gel preparation, including step：A certain amount of carbomer is added
Enter the dissolution of immune cell expansion suspension, pH value is adjusted to 6-6.5 using the sodium hydroxide solution of 0.1M, is slowly uniformly stirred in the process
It mixes.It is filled into prefabricated PE injection packaging, seals, packaging is stored in 4-10 DEG C.
In a kind of embodiment, the drug is that external application infiltrates preparation.
In a kind of embodiment, the main pharmacodynamics ingredient of external application infiltration preparation is：Immune cell expansion suspension.
In a kind of embodiment, sterile medical cotton ball is infiltrated on the immune cell expansion suspension, forms external application infiltration system
Agent.
In use, the sterile medical cotton ball that will infiltrate the immune cell expansion suspension, that is, external application infiltrate preparation
Directly smear or be covered in patient's uterus neck.
The second aspect of the present invention provides and a kind of treats HPV infection or HPV infection causes the method for disease, including step：
The uterus neck of patient will be covered or is applied to using immune cell expansion suspension as the drug of main pharmacodynamics ingredient.
In a kind of embodiment, external application infiltration system can be by the drug of main pharmacodynamics ingredient of immune cell expansion suspension
Agent.
In a kind of embodiment, sterile medical cotton ball is infiltrated on the immune cell expansion suspension, forms external application infiltration system
Agent.
In a kind of embodiment, immune cell expansion suspension (that is, suspension that acquisition is centrifuged after immune cell expansion) can lead to
The mode for crossing medical cotton ball infiltration, directly smears or is covered in patient's uterus neck.
In a kind of embodiment, external-use gel system can be by the drug of main pharmacodynamics ingredient of immune cell expansion suspension
Agent.
In a kind of embodiment, based on immune cell expansion suspension (that is, the suspension for being centrifuged acquisition after immune cell expansion)
The external-use gel preparation for wanting effective component to be prepared into is applied directly to patient's uterus neck.
It in a kind of embodiment, can be smeared every certain time primary, every course for the treatment of includes a few days, continuous multiple courses for the treatment of.Specifically
Suitable medicine-feeding method can be selected according to disease weight.For example, for HPV positive infection, can the next day smear primary, every course for the treatment of
15 days, continuous 3 courses for the treatment of.For the disease due to caused by HPV persistent infection：CIN or condyloma acuminatum etc. can smear one daily
It is secondary, every course for the treatment of 15 days, continuous 3 courses for the treatment of.
The therapy mechanism of the method is：Containing a large amount of immunocyte in amplification cultivation mistake in immune cell expansion suspension
Secreted a large amount of such as interferon, cell necrosis factor in journey, disposable cell amplification suspension act on patient's uterus neck, can
Achieve the purpose that Other diseases caused by treating HPV infection and further control and treating due to infection by passive immunity.
The third aspect of the present invention provides and a kind of treats HPV infection or HPV infection causes the drug of disease, main pharmacodynamics
Ingredient is immune cell expansion suspension.
In a kind of embodiment, the HPV infection refers to HPV test positive.
In a kind of embodiment, disease caused by the HPV infection refers to disease caused by HPV persistent infection.
In a kind of embodiment, disease caused by HPV persistent infection is that uterine neck tumor sample becomes (CIN), condyloma acuminatum or uterine neck
Cancer.
In a kind of embodiment, the drug is external preparation.
In a kind of embodiment, the drug is that preparation is smeared in external application.
In a kind of embodiment, the drug is exterior-applied gel.
In a kind of embodiment, the main pharmacodynamics ingredient of the external-use gel preparation is：Immune cell expansion suspension.
In a kind of embodiment, in the external-use gel preparation other than main pharmacodynamics ingredient, also containing can pharmaceutically connect
The auxiliary material received.These auxiliary materials can be the customary adjuvant for external-use gel preparation, such as carbomer.The dosage of auxiliary material, ability
Field technique personnel can determine according to actual needs.For example, the 3-5% (mass percent) of entire external-use gel preparation can be accounted for.
In a kind of embodiment, the preparation method of the external-use gel preparation, including step：A certain amount of carbomer is added
Enter the dissolution of immune cell expansion suspension, pH value is adjusted to 6-6.5 using the sodium hydroxide solution of 0.1M, is slowly uniformly stirred in the process
It mixes.It is filled into prefabricated PE injection packaging, seals, packaging is stored in 4-10 DEG C.
In a kind of embodiment, the drug is that external application infiltrates preparation.
In a kind of embodiment, the main pharmacodynamics ingredient of external application infiltration preparation is：Immune cell expansion suspension.
In a kind of embodiment, sterile medical cotton ball is infiltrated on the immune cell expansion suspension, forms external application infiltration system
Agent.
In use, the sterile medical cotton ball that will infiltrate the immune cell expansion suspension, that is, external application infiltrate preparation
Directly smear or be covered in patient's uterus neck.
Compared with prior art, the present invention has the advantages that：
Present invention firstly discovers that the use of immune cell expansion suspension being that main pharmacodynamics ingredient is applied to uterus neck, main
Isolation and isolation further infection touch opportunity are taken, HPV infection or the disease due to caused by HPV persistent infection can be treated,
HPV positive patient is set to turn out cloudy.By the positive patient for the HPV infection that the method is treated, after 7-8 treatment, negative conversion rate reaches
To 50% or more, negative conversion rate can reach 90% or more after 15 to 20 treatments, and can be effectively controlled and treat HPV and cause
Uterine neck tumor sample become (CIN) and cervical cancer relapse or transfer, achieve the effect that prevent and treat cervical carcinoma.So far, by exempting from
Epidemic disease cell correlation external application smearing makes HPV persistent infection quickly turn out cloudy and prevent and treat the method for cervical carcinoma both at home and abroad without report
Road, the method are pioneering both at home and abroad.
Specific embodiment
Therapy mechanism of the invention is：By immunocyte incubation secretion largely as interferon, meronecrosis because
Son etc., while immunocyte actively kills after contacting infected cell, at the same secrete cytokines killing infected it is thin
Born of the same parents, and further mediate out antibody by antigenic stimulus reach treatment HPV infection and into one by being actively and passively immunized jointly
The purpose of Other diseases caused by step is controlled and treated due to infection.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.The test method of actual conditions is not specified in the following example,
Usually according to normal condition, or according to condition proposed by each manufacturer.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Experimental material and its source of the invention：
1, experimental situation：It is operated in Biohazard Safety Equipment in the laboratory GMP；10000 grades of clean area, Biohazard Safety Equipment
100 grades.
2, reagent and consumptive material：
Interleukin-22 (the double aigret medicine companies in Beijing, 1,000,000 IU/ bottles), lymphocyte serum X-VIVO15 (Lonza),
CD16 monoclonal antibody (U.S. company BD), IL-21-K562 trophocyte's (Nanjing Suning health)；T75 Tissue Culture Flask
(NUCK), T175 Tissue Culture Flask (NUCK), cell culture bags GT-T610A (Takara) and pipette (NUCK).
3, used equipment includes when the embodiment of the present invention is tested：
The desk-top high-performance centrifugation of Biohazard Safety Equipment (Thermo), Thermo Scientific Sorvall ST16/16R
Machine, carbon dioxide incubator (Thermo, 3111), inverted biologic microscope (Motic, AE-31), automated cell calculating instrument
(Invitrogen, Countess), pipettor (Thermo) and GE WAVE wave bioreactor, peristaltic pump and ultrafiltration membrane packet
(10kd, 40kd) and fixture (PALL).
The infiltration preparation of 1 immune cell expansion suspension of embodiment and its preparation and application
The NK cell that the present embodiment is selected in immunocyte carries out amplification cultivation, separation, and supernatant is that NK cell expands suspension
(suspension of acquisition is also referred to as centrifuged after the amplification of NK cell), lower confluent monolayer cells are the NK cell after amplification.The NK cell expands
Increasing suspension can be used as main pharmacodynamics ingredient.NK cell after the amplification also can be used as main pharmacodynamics ingredient.
Specifically, the preparation method of the NK cell after NK cell amplification suspension, amplification includes the following steps：
(1) monocyte acquisition and acquisition：
Can be used singly adopt, after peripheral blood, placental blood, umbilical cord blood collection lymphocyte separation medium separation or separation of lymphocytes
Pipe separation obtains monocyte.
It is peripheral blood mononuclear cells used in the present embodiment；
(2) monocyte Fiber differentiation and NK cell expand：
Take peripheral blood mononuclear cells 4.0 × 107A carry out cell culture is stimulated using cell factor combination trophoderm and is induced
The method of culture, is inoculated in T75 square vase, and density is 1.0-1.5 × 106A cell/mL, culture medium based on X-VIVO15,
Wherein trophocyte 1.0 × 105A cell/mL, IL-2 100U/ml, IL12 20ng/ml according to culture amplification situation, by
Step expands culture, and fluid infusion is transferred to T175 culture, until 2L culture bag.
Reach within 13-14 days 2L cultivating system after the amplification of NK cell, and cell density reaches 2.0 × 106A cell/mL with
On, NK cell purity is greater than 90%, is centrifuged 15 minutes through 400G, collects supernatant respectively and lower confluent monolayer cells, gained supernatant are
For suspension after the amplification of NK cell.
The NK cell amplification suspension of acquisition is main pharmacodynamics ingredient, can be prepared into infiltration preparation.It is infiltrated by medical cotton ball
Mode, directly smear or be covered in patient's uterus neck.
Following step is specifically included using above-mentioned NK cell amplification suspension as the preparation method of the infiltration preparation of main pharmacodynamics ingredient
Suddenly：
The NK cell of above-mentioned acquisition is expanded into suspension, intermediate product are obtained after 40kd is filtered, it is common with sterile medical cotton ball
It is put into 4-10 DEG C of storage in the medical sealing container of 50ml, as infiltration preparation.
It can be used directly after taking-up.
NK cell expands the external application that suspension is main pharmacodynamics ingredient and infiltrates preparation clinical application controlling in HPV persistent infection
It treats：
Treatment object：Volunteer, 21 altogether, be women, and the age is between 20~50 years old, clinically to have suffered from
The crowd of HPV persistent infection.
Therapeutic modality：It is daily or the next day cervical department of volunteer is applied directly to using cotton balls infiltration NK cell amplification suspension
Position；
Therapeutic effect judgement：It is negative that round pcr detects HPV-DNA.
Treatment results statistics：Altogether in 21 patients, after the treatment method treatment in the present embodiment, wherein 2 people are not
Check, 5 people are still positive.Using the positive patient of the HPV infection of the treatment method treatment in the present embodiment, treated through 7-8 times
Afterwards, negative conversion rate reaches 31.8% or more.Negative conversion rate can reach 52.6% or more after 15 to 20 treatments.Pass through follow up
Know, can be effectively controlled and treat uterine neck tumor sample caused by HPV and become (CIN) and cervical cancer relapse or transfer, reach prevention and
The effect for treating cervical carcinoma.
Typical case：
1, volunteer 1-1, the age 44 years old, medical history：In October, 2016,52 types of cervical carcinoma screening hpv were positive, in January, 2017
Cervical biopsy：Normally, check hpv52 type on July 21st, 2017 is positive；Therapeutic modality：2017.9.15-9.27 the next day palace
Neck smears the external application infiltration preparation of the amplification suspension of the NK cell in the present embodiment；Index observing：Leukorrhea normal flora is normal；One
Month review result：The HPV positive (2017.10.13)；Three months review results：HPV feminine gender (2017.11.17).
2, volunteer 1-2, the age 32 years old, medical history：10 cervical carcinoma screening hpv are positive within 2016；On 09 8th, 2017 uterine neck
TCT：Normally, HPV82 is positive；Therapeutic modality：2017.9.15-9.27 the next day, uterine neck smeared the NK cell in the present embodiment
The external application for expanding suspension infiltrates preparation；Index observing：Leukorrhea white secretion flora is normal；It checks within one month：HPV is positive
(2017.10.12)；Check (2017.11.15) in three months.
3, volunteer 1-3, the age 46 years old, medical history：Participation cervix cancer in China screening hpv is positive within 2016 10；In January, 2017
Cervical biopsy：Normally；2017.09.26 hpv23 parting is checked：Low danger 44 is positive；Treatment method：2017.9.19-9.25 daily
Uterine neck smears the external application infiltration preparation of the present embodiment NK cell amplification suspension；Index observing：Leukorrhea normal flora is normal；One
It checks within a month：HPV feminine gender (2017.10.12)；It checks within three months：HPV feminine gender (2017.11.15).
4, volunteer 1-4, the age 42 years old, medical history：Participation cervix cancer in China screening hpv is positive within 2016 10；Non- row vagina
Mirror+biopsy, does not go any processing.08 month 2017 25 uterine neck TCT：Normally；Biopsy：Normally；Check hpv23 parting：High-risk 18,
66,68 is positive, and low danger 6,11 is positive；Treatment method：2017.9.15-9.27 the next day, uterine neck smeared the present embodiment NK cell
The external application for expanding suspension infiltrates preparation；Index observing：Leukorrhea normal flora is normal；It checks within one month：HPV is negative
(2017.10.13)；It checks within three months：HPV feminine gender (2017.11.13).
5, volunteer 1-5, the age 35 years old, medical history：2017.4.13 uterine neck TCT：Atypical cell；2017.9.26 check
Hpv23 parting：Low danger 43 is positive；Treatment method：2017.9.19-9.25 uterine neck smears the expansion of the present embodiment NK cell once a day
The external application for increasing suspension infiltrates preparation；Index observing：Leukorrhea normal flora is normal；It checks within one month：HPV is positive
(2017.10.12)；It checks within three months：HPV feminine gender (2017.11.13).
The infiltration preparation of 2 immune cell expansion suspension of embodiment and its preparation and application
The NK cell that the present embodiment is selected in immunocyte carries out amplification cultivation, separation, and supernatant is that NK cell expands suspension
(suspension of acquisition is also referred to as centrifuged after the amplification of NK cell), lower confluent monolayer cells are the NK cell after amplification.The NK cell expands
Increasing suspension can be used as main pharmacodynamics ingredient.NK cell after the amplification also can be used as main pharmacodynamics ingredient.
Specifically, the preparation method of the NK cell after NK cell amplification suspension, amplification includes the following steps：
(1) monocyte acquisition and acquisition：
Can be used singly adopt, after peripheral blood, placental blood, umbilical cord blood collection lymphocyte separation medium separation or separation of lymphocytes
Pipe separation obtains monocyte.
It is peripheral blood mononuclear cells used in the present embodiment；
(2) monocyte Fiber differentiation and NK cell expand：
Take peripheral blood mononuclear cells 4.0 × 107A carry out cell culture is stimulated using cell factor combination trophoderm and is induced
The method of culture, is inoculated in T75 square vase, and density is 1.0-1.5 × 106A cell/mL, culture medium based on X-VIVO15,
Wherein trophocyte 1.0 × 105A cell/mL, IL-2 100U/ml, IL12 20ng/ml according to culture amplification situation, by
Step expands culture, and fluid infusion is transferred to T175 culture, until 2L culture bag.
Reach within 13-14 days 2L cultivating system after the amplification of NK cell, and cell density reaches 2.0 × 106A cell/mL with
On, NK cell purity is greater than 90%, is centrifuged 15 minutes through 400G, collects supernatant respectively and lower confluent monolayer cells, gained supernatant are
For suspension after the amplification of NK cell.
The NK cell amplification suspension of acquisition is main pharmacodynamics ingredient, can be prepared into external-use gel preparation and carries out outside uterine neck smearing
With.
Using above-mentioned NK cell amplification suspension as the preparation method of the external-use gel preparation of main pharmacodynamics ingredient specifically include as
Lower step：
The NK cell of above-mentioned acquisition is expanded into suspension, intermediate product are obtained after 40kd is filtered, then by a certain amount of card wave
The dissolution of immune cell expansion suspension is added in nurse, slowly equal in the process using the sodium hydroxide solution adjusting pH value of 0.1M to 6-6.5
Even stirring.It is filled into prefabricated PE injection packaging, seals, packaging is stored in 4-10 DEG C.
The external-use gel preparation clinical application of immune cell expansion suspension is in the treatment of HPV persistent infection：
Treatment object：Volunteer, 15 altogether, be women, and the age is 20~50 years old, is held clinically to have suffered from HPV
The crowd of continuous infection.
Treatment method：It is daily or the next day to use NK cell amplification suspension be the external-use gel preparation smearing of main pharmacodynamics ingredient
In the uterus neck of volunteer
Therapeutic effect judgement：It turns out cloudy and refers to that round pcr is that detection HPV-DNA is negative.
Negative conversion rate refers to the percentage of turn out cloudy patient populations and participation experimenter's quantity.
Treatment results statistics：Altogether in 15 patients, after the treatment method treatment in the present embodiment, wherein 1 people is not
Check, 6 people check that, to be positive, the positive patient of HPV infection, after 7-8 treatment, negative conversion rate reaches 33.3% or more twice.
Negative conversion rate can reach 53.3% or more after 15 to 20 treatments.Known by follow up, can be effectively controlled and treat HPV
Caused uterine neck tumor sample becomes (CIN) and cervical cancer relapse or transfer, achievees the effect that prevent and treat cervical carcinoma.
Typical case：
1, volunteer 2-1, the age 39 years old, medical history：Check that hpv18 type is positive in November, 2016；On July 28th, 2017 uterine neck
TCT：Normally, HPV39 is positive；Therapeutic modality：2017.9.20-9.27 uterine neck smears the amplification of the present embodiment NK cell once a day
Suspension is the external-use gel preparation of main pharmacodynamics ingredient；Index observing：Leukorrhea normal flora is normal；One month review result：HPV
Negative (2017.10.20)；Three months review results：HPV feminine gender (2017-11.17).
2, volunteer 2-2, the age 29 years old, medical history：Look on September 20th, 2017 high-risk 52, the 68 type positive of hpv, 81 sun of low danger
Property, it is doubtful high-risk 73 positive；Therapeutic modality：2017.9.15-9.27 uterine neck was smeared the present embodiment NK cell and was expanded and hangs the next day
Liquid is the external-use gel preparation of main pharmacodynamics ingredient；Index observing：Leukorrhea normal flora is normal；One month review result：It is not multiple
It looks into；Three months review results：HPV feminine gender (2017-11.15).
3, volunteer 2-3, age：42 years old；Medical history：08 cervical carcinoma screening hpv16 type is positive within 2016；01 moon palace in 2017
Neck TCT：Normally；Treatment method：2017.9.20-9.26 uterine neck is smeared based on the present embodiment NK cell amplification suspension once a day
Want the external-use gel preparation of effective component；Index observing：Leukorrhea normal flora is normal；One month review result：HPV is positive
(2017.10.12)；Three months review results：HPV feminine gender (2017.11.15).
4, volunteer 2-4, the age 34 years old, medical history：2017.09.14hpv high-risk 51, the 58 types positive；Treatment method：
2017.9.15-10.13 the next day, uterine neck smeared the external-use gel that the present embodiment NK cell amplification suspension is main pharmacodynamics ingredient
Preparation；Index observing：Leukorrhea normal flora is normal；It checks within one month：HPV feminine gender (2017.10.13)；It checks within three months：HPV
Negative (2017.11.13).
5, volunteer 2-5, the age 45 years old, medical history：2016.10 it is positive to participate in cervix cancer in China screening hpv；2017.01 just
Often；Intravaginal drug, specific medication are unknown；2017.09.5 checking 23 parting of hpv：High-risk 52,58 is positive；Low danger 81 is positive；It controls
Treatment method：2017.9.20-9.26 the suspension of the present embodiment NK cell amplification once a day is the external-use gel system of main pharmacodynamics ingredient
Agent；Index observing：Leukorrhea normal flora is normal；It checks within one month：The HPV positive (2017.10.12)；It checks within three months：HPV yin
Property (2017.11.13).
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that under the premise of not departing from the method for the present invention, can also be made for those skilled in the art
Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are equivalent embodiment of the invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical solution of the present invention
It is interior.
Claims (18)
1. the purposes that immune cell expansion suspension is used to prepare the drug of disease caused by treatment HPV infection or HPV infection.
2. purposes according to claim 1, which is characterized in that the immune cell expansion suspension is main pharmacodynamics ingredient.
3. purposes according to claim 1, which is characterized in that the immune cell expansion suspension refers to immunocyte through expanding
Increase the supernatant obtained after culture, separation.
4. purposes according to claim 1, which is characterized in that after amplification cultivation, immunocyte density reaches 2.0 × 106It is a
Cell/mL or more；Preferably, after amplification cultivation, immunocyte density reaches 3.0 × 106A cell/mL or more；More preferably,
After amplification cultivation, immunocyte density reaches 4.0 × 106A cell/mL or more.
5. purposes according to claim 1, which is characterized in that used method is selected from when the amplification：Cell factor
Stimulus method, antibody stimulating method or trophocyte's amplification.
6. purposes according to claim 1, which is characterized in that the immune cell expansion suspension is that the amplification of NK cell is outstanding
Liquid, the NK cell amplification suspension refer to the supernatant that NK cell obtains after amplification cultivation, separation.
7. purposes according to claim 1, which is characterized in that NK cell is obtained by monocyte culture；Further, institute
Cells of monocytic origin is stated in peripheral blood, Cord blood or placental blood.
8. purposes according to claim 1, which is characterized in that the HPV infection refers to HPV test positive.
9. purposes according to claim 1, which is characterized in that disease caused by the HPV infection refers to HPV persistent infection
Caused disease.
10. purposes according to claim 9, which is characterized in that disease caused by HPV persistent infection be uterine neck tumor sample become,
Condyloma acuminatum or cervical carcinoma.
11. purposes according to claim 1, which is characterized in that the drug is external preparation, further, the medicine
Object is that preparation is smeared in external application.
12. purposes according to claim 11, which is characterized in that the drug is exterior-applied gel or external application infiltration system
Agent.
13. purposes according to claim 11, which is characterized in that the exterior-applied gel contains immune cell expansion suspension
With pharmaceutically acceptable carrier auxiliary material；The external application infiltration preparation includes immune cell expansion suspension and has infiltrated immunocyte
Expand the sterile medical cotton ball of suspension.
HPV infection is treated or HPV infection causes the method for disease, including step 14. a kind of：To be with immune cell expansion suspension
The drug of main pharmacodynamics ingredient covers or is applied to the uterus neck of patient.
HPV infection is treated or HPV infection causes the drug of disease 15. a kind of, and main pharmacodynamics ingredient is immune cell expansion suspension.
16. drug according to claim 15, which is characterized in that the drug is external preparation, further, the medicine
Object is that preparation is smeared in external application.
17. drug according to claim 15, which is characterized in that the drug is exterior-applied gel or external application infiltration system
Agent.
18. drug according to claim 15, which is characterized in that the exterior-applied gel contains immune cell expansion suspension
With pharmaceutically acceptable carrier auxiliary material；The external application infiltration preparation includes immune cell expansion suspension and has infiltrated immunocyte
Expand the sterile medical cotton ball of suspension.
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| CN201810614761.4A CN108815186A (en) | 2018-06-14 | 2018-06-14 | A kind of drug and preparation method and application treated HPV persistent infection and cause disease |
| PCT/CN2019/091260 WO2019238120A1 (en) | 2018-06-14 | 2019-06-14 | Medicament for treating diseases caused by persistent hpv infection, preparation method therefor and use thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019238120A1 (en) * | 2018-06-14 | 2019-12-19 | 上海鸣大生物科技有限公司 | Medicament for treating diseases caused by persistent hpv infection, preparation method therefor and use thereof |
| CN113318126A (en) * | 2021-06-15 | 2021-08-31 | 海南启研干细胞抗衰老医院有限公司 | Technology for treating HPV infected patient |
| WO2024087356A1 (en) * | 2022-10-28 | 2024-05-02 | 上海市东方医院（同济大学附属东方医院） | Use of mesenchymal stem cells in preparation of drugs for treating persistent hpv infection |
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| CN113318126A (en) * | 2021-06-15 | 2021-08-31 | 海南启研干细胞抗衰老医院有限公司 | Technology for treating HPV infected patient |
| WO2024087356A1 (en) * | 2022-10-28 | 2024-05-02 | 上海市东方医院（同济大学附属东方医院） | Use of mesenchymal stem cells in preparation of drugs for treating persistent hpv infection |
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| WO2019238120A1 (en) | 2019-12-19 |
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Application publication date: 20181116 |