Biotransplant and method for treating mental development delay cases

FIELD: medicine.

SUBSTANCE: method involves introducing proper mesenchyme stem cells separated from marrow and cultivated in vitro. The cells are implanted on a carrier into cerebral cortex region planned in advance. To do it, the mesenchyme stem cells are preliminarily introduced into modified resorbable stent (carrier). The number of cells to be introduced is equal to 7-12 mln per one Brodman zone selected for transplanting autologic mesenchyme stem cells. Marked anatomical changes in brain regions being the case, the same quantity of cells are inravascularly (intravenously or intra-arterially) introduced. The treatment is carried out in particular cases in concurrently introducing the mesenchyme stem cells in intraperenchymatous or intravenous way.

EFFECT: enhanced effectiveness of treatment in cognitive-mnestic function disorder cases.

10 cl

 

The invention relates to medicine, namely to psychiatry, and can be used in clinical psychiatry in the treatment of lesions of the intellectual-mnestic functions of various etiologies. This large group of disorders include severe forms of mental retardation.

Mental retardation children registered in all countries, the statistics are about 0,25-0,50% of the population with intellectual coefficient (IQ) less than 50, which is indicative of a severe and pronounced mental retardation. The prognosis adversely. Congenital dementia people has two mandatory attributes: 1) General mental underdevelopment with the prevalence of intellectual disability; 2) the lack of progression, indicating pathological process (Sukharev G. Lectures on psychiatry of childhood. - M.: Medicine. 1974 - 320 C.).

One of the features of the pathogenesis of mental retardation is that malformation (disinfopedia) occurs due to damage to the body, not yet completed development. He is fundamentally different from the defect is due to damage to an already formed body.

The clinical picture of dysontogenetic, including congenital anomalies, mental development differs significantly from clinical lesions formed organs and systems. In the first case, there is raised a developmental disorder and the second acquired defect normal to the functions. The main feature of the diffuse "total" underdevelopment is a violation of the psyche as a whole.

It is noted that in patients showing signs of underdevelopment of intelligence, thinking, and other mental functions (perception, memory, attention, language, motor skills, emotional-volitional sphere and so on).

Picture of lesions depends not only on the nature and intensity of damage, but on the features of the period of ontogenesis, which affected developing body and the organism as a whole, i.e. from chromogenes factor. Depending on the depth of mental underdevelopment allocate 3 degrees of mental retardation: the idiocy (IQ<20), imbecile (IQ=20-50), debilnosti (IQ 50-70).

Treatment of lesions of the intellectual-mnestic functions associated with effects on the Central nervous system (CNS), the brain cortex. There is a method of treatment of such disorders by neuroprotective, resolving, dehydration, vitamin and restorative therapy (Manual on psychiatry./ Edited Avinasha. - M.: Medicine, 1983, Vol.2, s-507 - [1]).

Also known a method of neurotransplantation embryonic stem cells (NESK), which is used in various treatment-resistant CNS lesions (Ashbrooke. Transpl stacia nerve cells and tissue engineering of brain and nervous diseases. - M., 2003, s, Semchenko V.V. and other Neurotransplantation, Omsk, 2004, s. - [2, 3]).

One of the most modern and actively develop ways to manage treatment-resistant, lesions of the Central nervous system (CNS) is intracranial transplantation of mesenchymal stem cells (MSCS). This is a promising method to restore disturbed functions of the Central nervous system, in which, besides the possibility of purposeful influence on certain functional structure of the brain, lies a normalizing effect on the imbalance of the neurotransmitter systems of the brain, which is actually a fundamentally new approach to the treatment of a large group of diseases associated with damage to the Central nervous system.

In contrast to the use in neurotransplantation as donor material embryonic tissues, in particular embryonic neuroblasts person obtained from the bone marrow of the patient MSK (autologous transplantation), can get rid of many problems.

First of all, completely removed the problem of the blood-brain and immune responses, as are transplanted to the patient's own cells. In this autoimmune cells not only cause rejection reactions, but also possess immunosuppressive properties.

When employing the autotransplantation M Is To disappear religious, ethical and legal problems associated with the use of allogenic neuroblasts, which greatly complicate the use of embryonic tissue or cell cultures derived from them.

In addition, significantly simplified organizational problems associated with the need (for neurotransplantation embryonic neuroblasts) survey of donor tissue for many viral infections and genetic diseases.

MSC are cells-precursors, which are then further specialization are formed or nerve cells, or cells of various tissues. "Specialization" MSC depends on the immediate cellular environment in which falls MSC. Thus, MSC transplanted directly into the brain tissue or introduced into the brain through the vascular system becomes functional features of the surrounding neurons. In the future, some of them catch on, differentiated and establishes communication with the surrounding nerve cells. Provided by them to the imbalance of the neurotransmitter systems of the patient brain normalizing effect is a fundamentally new approach in the treatment of many diseases associated with damage to the Central nervous system.

Transplantation of MSC in the cerebral cortex related to their ability to improve the function of immature (hit) of the brain, to form a connection in certain areas of the CNS due to the synthesis of neurotransmitters, which may contribute to the development of mental and behavioral abilities. Autologous MSCS have advantages over the donor MSC in the transplantation takes place without complications and immune responses. As the carrier MSK use pharmaceutically acceptable hemostatic sponge, which dissolve with time, performing its function.

Brain development is also facilitated the introduction of autologous MSC vnutrisosudisto (intravenously or intraarterially). This MSC transplantation into the brain flows through the circulatory system of the brain: the blood MSCS migrated to the lesion. Tested also the simultaneous introduction of autologous MSC: by intravascular injection and intracortical transplantation in the brain. In this case, is a more complete saturation of MSC cells of the cerebral cortex, providing them the lesion.

The closest in technical essence and the achieved result claimed is a method of treatment delay, mental and intellectual development of different origin by neurotransplantation embryonic nervous tissue in the brain cortex (RU, patent No. 2219937, 2003).

The technical problem on which the present invention is directed, the conclusion is raised to increase the effectiveness of treatment reduction expressed intellectual-mnestic functions, associated with mental underdevelopment, with the breaking of these links the Central nervous system. The treatment is carried out by transplantation of autologous mesenchymal stem cells in different parts of the cerebral cortex, functional features which, to the maximum extent associated with the existing pathology, and/or intravascular introduction of GMT.

The bone marrow of the patient to obtain autologous MSC were taken by puncture under short-term anesthesia from the sternum or the wing of the Ilium of the pelvis. Then punctal were treated with heparin, diluted nutrient medium, centrifuged, MSC were isolated and cultured them in the growth medium containing nutrient medium Needle in the modification Dulbecco (DMEM), resulting in at 87-99% homogeneous phenotype living cells.

As the nutrient medium when working with autologous cells was selected nutrient medium, DMEM, which allowed stable, with a high growth rate to obtain highly homogeneous MSC sufficient for transplantation quantity. Environment DMEM available due to its prevalence and universality, but depending on the quality and source of seed cellular material, its composition usually modify. In the invention the environment DMEM used without any additions and amendments, as by centrifugation were you the Helena viable MSC high degree of homogeneity. The use of such cells as inoculum on the growth environment, using DMEM were obtained for biotransplant cells and their number is ten times higher than the content in the bone marrow. After passage and removal of the mattresses was obtained concentrate, containing MSC 8-10×106in 1 ml.

Biotransplant for the treatment of mental retardation is a suspension of cells in 10% autologous serum in physiological solution containing autologous mesenchymal stem cells (MSC), a homogeneous cell markers CD 44+, CD 90+, CD 105+, CD 106+, CD 45-CD 34-. Instead of 10% autologous serum in saline solution (solvent suspension) you can use other solvents selected from pharmaceutically acceptable: autoplasma in saline solution, autoplasma, autoseparate in any % from 5 to 100, and glucose 10%, 5%, 7% saline solution. In 1 ml of biotransplant contains 0,7-1,2×106MSK.

For neurotransplantation using autologous mesenchymal stem cells derived from the bone marrow of the patient and further cultured in vitro. Cells injected into the affected area, i.e. the lung, in view of the above suspension. Treatment of cells injected vnutrisosudisto: V/a - intraarterially,/ which - intravenously, and the total number of input MSK is from 7 to 25 million

For holding neurotransplantation use biotransplant containing 40-45 million young MSC with a concentration of 0.7 to 1.2×106in 1 ml of biotransplant, introducing a modified stent, which is their medium. As the carrier preferably used hemostatic sponge. Standard hemostatic sponge 9×9 usually use 20 ml of cell suspension. Fragments of saturated MSC stent with the number of MSC 7-12×10 per area implanted in one or more of the predefined areas of the cerebral cortex. It is, first of all, the realm of the functional fields of Brodman 44, 43, 46, 24, 25, 22, 41 and 42. Further, the stent undergoes gradual resorption, and were in MSC washed away by the blood stream and is integrated into the parenchyma of the brain. In some cases, to achieve a better effect, are applied simultaneously intraparenchymatous and intravascular routes of administration MSC, i.e. when the operation combine both methods of cell injection, and the additional introduction is carried out by a dosage of 0.5-0.7×106autologous mesenchymal stem cells per 1 kg of patient's weight.

Since the time of autologous MSCS for use as biotransplant course of time, you may experience the sieve is tion, when the operating time (accumulation) of cells produced, and the patient's health is not ready for treatment. In such cases, the cellular material is subjected to cryopreservation, which saves MSC unchanged. After defrosting cells have all their properties have the same characteristics of the phenotype and remain alive. Cryopreservation was performed using as a cryoprotectant dimethyl sulfoxide, as it is pharmaceutically acceptable and compatible with the used solvents in suspension introduced in the treatment of autologous cells.

Biotransplant after cryopreservation has been used in patients as young cells described above. The treatment results are comparable with the use of freshly prepared biotransplant.

When anatomical changes (or absence) of functionally important areas of the brain MSK enter only vnutrisosudisto (V/a - at the rate of 2 ml/min or in/at the speed of 6 ml/min), and the total number of input MSK is from 7 to 25 million the Number of MSC is calculated from 0,5-0,7×106cells per 1 kg of patient's weight. For transplantation were used freshly prepared cells, if the treatment was made with them, or christopherovna MSC, if the treatment they participated.

One of the important conditions is the reduction of lying to the frame between the extraction MSK from cultivated environment and transplanted directly into the brain of the patient. This is because in every minute of this period a certain number of cells becomes unsustainable, and this ultimately affects the final effect. The method is illustrated on the example of its concrete implementation.

Example 1:

Patient K., born in 1981, № history 1836. Pregnancy the mother proceeded with morning sickness, childbirth with severe asphyxia. The first 7 days of life the patient was in the intensive care unit. Early development delay, sat in 1.5 years, independently went in 5 years. Pronounced delay speech development in early childhood. The patient was continuously treated with various drugs as outpatients and more than 12 times in different hospitals. A learning disability, an improvement in the condition of the patient was not recorded.

Upon admission to the ward (age 24 years) patient productive contact is not available, almost no servicing. Turned it partially understands. The number of correctly used words does not exceed 3-8, properly executed teams does not exceed 5-7. Speech slurred. The IQ level corresponds mental retardation in the degree of idiocy (<20). Pronounced syndrome of motor hyperactivity with destructive tendencies, attention is held no more than 2-3 seconds. The patient needs constant care and supervision.

Due to the ineffectiveness and Besparmak what ewnetu medical treatment were considered the question of holding neurotransplantation. After the positive opinion of the ethics Committee and obtaining written consent of the relatives, it was decided to conduct intraparenchymatous autologous transplantation of mesenchymal stem cells cultured from bone marrow of the patient.

During surgery bilaterally in the frontal region (projection point Kocher) was intraparenchymatous introduced by 7.5 million cells and vnutrisosudisto 7 million cells. The surgery went without complications, the sutures were removed on the 8th day.

Already in the first days after surgery significantly decreased effects of motor disinhibition, almost completely disappeared aggressiveness. It was noted the positive dynamics in the speech-motor and emotional spheres. After 0.5 years after autologous transplantation of MSC supply correctly used words of more than 30, the number of correctly executed commands about 80. The patient fully itself serves, dresses, undresses, and removes Telit bed, independently uses the toilet. Can independently brew coffee, to clear the table, etc. The patient appeared capacity for learning. It is with pleasure for hours considering (and correctly names) picture books, watching TV, playing with dolls. However, the correct, targeted actions are sick only in respect of "interesting" for her actions. For the provide to do anything against her desire extremely problematic. Upon further observation of the patient's condition continues to progressively improve.

Example No. 2:

Patient S year of birth, № history - 2584. Heredity is not burdened. Pregnancy the mother proceeded with toxicity and placental abruption. Births preterm (34 weeks), caesarean section. One hour after birth, the child has developed asphyxia, and he was transferred to the intensive care unit, where he remained on a ventilator for months. In a further development with a delay. Head held 6 months, villages in 11 months, alone stood at 13. Prattling speech with 1.5 years, single words and short phrases to 3 years. In the future completely stopped talking, turned it partially understood. From birth was seen by a neurologist, had been diagnosed with perinatal encephalopathy syndrome musculoskeletal disorders. Regularly takes medication. From the age of 6 months. Regularly treated in different hospitals (17 hospitalizations), with different diagnoses: cerebral palsy (spastic-asthenic form), to the syndrome of early infantile autism (cerebral organic origin). Last year visited the rehabilitation Center for the child" where he observed the reaction of negativity aggression against teachers and children. The patient tried to study the program of the 1st class, however, the program was not learned.

Admission: Comtat the t formal productive contact is not available, it is missing. In behavior inappropriate, inconsistent, attention is extremely unstable. Correctly perform 8-10 simple commands. Itself serves in part, unkempt. IQ is reduced to the level of idiocy, the capacity for learning is missing. In the absence of the effect of the treatment and the continuing deterioration of the patient it was decided to hold neurotransplantation. After a positive decision of the ethics Committee and obtaining written consent of the relatives were autologous mesenchymal stem cells. During autotransplantation bilaterally in the region of the prefrontal cortex the patient was introduced to 8 million cells and simultaneously vnutrisosudisto entered 53 million mesenchymal stem cells with a speed of 6 million cells per minute, intravenous injection.

In the first weeks after transplantation, the patient was significantly reduced aggressiveness towards others. In the future the patient began to pronounce correctly and use short words and phrases. Significantly expanded the scope of self-service, the patient started to use the toilet, dress-undress, clean bed, etc. Subsequent follow-up showed significant improvement in verbal and cognitive functions in a patient appeared pic the institutional capacity for learning.

Example No. 3:

Patient A. born in 1992. Pregnancy the mother proceeded with morning sickness, childbirth with severe asphyxia. Development delay. Noted progressive delay of speech development, aggressiveness in communication, unstable focus and the absence of memory in learning.

The IQ level<50. After the Commission's conclusions about the futility of medical treatment and obtain the written consent of the relatives had an operation on transplantation of autologous mesenchymal stem cells (MSCS). MSC isolated from bone marrow of the patient by processing the tissue to obtain a homogeneous phenotype of the cells, then cultured in vitro. The transplant was a modified stent, which is a media saturated MSC, the number of cells that are made in areas of the brain, amounted to a total of 42 million

During operation intraparenchymatous bilaterally in the frontal region introduced biotransplant. After 7 days the stitches were removed. Marked improvement of the patient in 2 weeks. Aggressiveness is not observed. Observation during the year confirmed the validity of the decision for surgery: the patient was teaching, increased vocabulary, a positive dynamics in the speech-motor and emotional spheres.

Example No. 4:

Patient Century. born in 1996. Pregnancy in mA is Yeri proceeded with toxicosis. Childbirth without features. With 2 years diagnosed with delayed speech development", then "stereotypie patient with developmental delay". At the age of 8 intellectual development correspond to the age of 2 years (embezelment). After obtaining the permission of the parents of the patient were vnutrisosudisto entered MSC. The introduction was carried out by intravenously at a speed of 6 million cells per minute and intraarterially at a speed of 2 m/min. MSC were autologous, they were obtained from the patient's bone marrow and cultured in vitro. The total number of introduced cells was 54 million Cells were uniform in phenotype and viable.

Observation of the patient during the first month after the introduction of the MSC showed a significant improvement of the patient's condition that manifested itself both in behavior and in the expression of interest when displaying images in books, sustained attention. Child observation continues, progress in speech development.

Example # 5:

Patient P. 1989 was born in the Department with a Diagnosis of cerebral palsy, spastic form with marked developmental delay (imbecile). 24.02.05. was extracted bone marrow with subsequent culturing of autologous mesenchymal stem cells and storing the obtained culture in the special conditions, the deep freeze. Again the hospitalization (11.04.05) finished MSC culture was thawed and transplanted patient by intravascular injection, moreover, the total number of input MSK amounted to 25 million cells, the introduction was carried out intraarterially at the rate of 2 million cells per minute.

Before transplantation, the condition was characterized by a complete absence of speech, partial self-service, total disinhibition, irritability, lack of ability to learning and need constant care and supervision. A year later, when admission control, showed a significant clinical improvement. The level of non-verbal intelligence was significantly increased (p<0.03 in). Even though it is not formed, the patient began to make some syllables and short words. Vocabulary reaches 50, the number of correctly executed commands >100. Can spend hours alone to play or watch TV. The patient appeared capacity for learning, it completely serves. Became fully take care of himself. Significant positive changes were derived from the social test (p<0,01).

The present invention improves the efficiency of treatment reduction expressed intellectual-mnestic functions associated with lesions of the Central nervous system.

Our proposed method was tested in psychoneurological research Institute. After academician V.M. bekhterov, at the Department of surgery of nervous and mental diseases.

The method of autotransplant is treated mesenchymal stem cells is a new method of treatment of patients with lesion of the intellectual-mnestic functions of various etiologies.

1. Biotransplant for the treatment of mental retardation, characterized by the fact that is a suspension of cells in 10%autologous serum in saline solution or in a pharmaceutically acceptable solvent, containing a homogeneous cell markers CD 44+, CD 90+, CD 105+, CD 106+, CD 45-CD 34-autologous mesenchymal stem cells (MSC)derived from bone marrow, for which the fabric desagregirutee heparin, the cell suspension is centrifuged, homogeneous MSC cultured in growth medium containing nutrient medium Needle in the modification Dulbecco.

2. Biotransplant according to claim 1, characterized in that it contains freshly prepared cell culture.

3. Biotransplant according to claim 1, characterized in that it contains a cell culture, cryopreserved using as a cryoprotectant dimethyl sulfoxide.

4. Biotransplant according to claim 2 or 3, characterized in that it contains 0,7-1,2·106MSC in 1 ml of biotransplant.

5. Biotransplant claim 2 or 3, characterized in that further comprises a pharmaceutically acceptable carrier at a concentration of MSC 0,7-1,2·106in 1 ml of biotransplant.

6. A method of treating patients with severe mental underdevelopment through the use of medical means, characterized in that as a remedy using Biot is ansplant according to claim 4 or 5, which enter the lung in the number of 7-12 million autologous mesenchymal stem cells on one of the zones of Brodman 44, 43, 46, 24, 25, 22, 41 and 42.

7. The method according to claim 6, characterized in that it further impose biotransplant according to claim 2 or 3 vnutrisosudisto with dosage of 0.5-0.7·106autologous mesenchymal stem cells per 1 kg of patient's weight.

8. A method of treating patients with severe mental underdevelopment through the use of medical means, characterized in that as a therapeutic tools use biotransplant according to claim 2 or 3, which is injected vnutrisosudisto, and the total number of input MSK is from 7 to 25 million

9. The method according to claim 7 or 8, characterized in that the intra-arterial injection of biotransplant carried out at the rate of 2 ml/min

10. The method according to claim 7 or 8, characterized in that the intravenous biotransplant carried out with a speed of 6 ml/min



 

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